Pharmaceutical Composition for Cancer Treatment

ABSTRACT

The present invention provides a pharmaceutical composition for cancer treatment comprising an antibody against CCR8.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition for cancertreatment comprising an antibody against CCR8.

BACKGROUND ART

Potent negative regulation mechanisms, including immunosuppression,mediated by regulatory T cells (Treg cells) in the tumormicroenvironment are major obstacles to the treatment of tumors (NonPatent Literature 1).

For example, CD4-positive Treg cells which infiltrate tumors may be ableto strongly inhibit antitumor immune response and may become a majorobstacle to effective cancer treatment.

Tumor immunosuppression mediated by CD4-positive FoxP3-positive Tregcells has been sufficiently demonstrated in animal tumor models. It hasbeen reported that systemic (including intratumoral) Treg cell removalproduces an antitumor effect, wherein the removal of approximately 50%tumor-infiltrating Treg cells is not effective (Non Patent Literature2).

It has been reported that the increased ratio of CD4-positiveCD25-positive Treg cells (cell population including Treg cells) to thewhole CD4-positive T cell population in humans is intratumorallydetected in patients with various cancers including lung, breast, andovary tumors, and the abundance ratio correlates negatively with thesurvival probabilities of the patients (Non Patent Literatures 3 to 8).

The removal of CD4-positive CD25-positive Treg cells from tumors usingan anti-CD25 antibody has been confirmed to produce an antitumor effect.However, this removal is not specific for the Treg cells because CD25 isexpressed on the cell surface of the CD4-positive CD25-positive Tregcells as well as newly activated effector T cells. Furthermore, theadministration of an anti-CD25 antibody to mice brings about a limitedantitumor effect. It has been demonstrated in various tumor models thatonly the antibody administration before tumor inoculation exhibits atherapeutic effect, whereas the administration of the antibody aftertumor engraftment in mice rarely produces a therapeutic effect. Theantitumor effect was attenuated in the case of starting theadministration of an anti-CD25 antibody at post-transplant day 1, andwas rarely observed in the case of starting the administration of ananti-CD25 antibody at post-transplant day 2 or later (Non PatentLiterature 9).

Drug efficacy tests have been carried out so far by administeringantibodies to mice for the purpose of removing Treg cells. Nonetheless,there are few reports showing an antitumor effect. Thus, it is verydifficult to confirm an antitumor therapeutic effect brought about byTreg cell removal by antibody administration before inoculation (NonPatent Literature 10).

CCR8, also previously called CY6, CKR-L1 or TER1, is a G protein-coupled7-transmembrane CC chemokine receptor protein expressed in the thymus,the spleen, etc. A gene encoding this protein resides on humanchromosome 3p21. Human CCR8 consists of 355 amino acids (Non PatentLiterature 11). CCL1 is known as an endogenous ligand for CCR8 (NonPatent Literature 12). Human CCR8 cDNA is constituted by the nucleotidesequence represented by GenBank ACC No. M_005201.3, and mouse CCR8 cDNAis constituted by the nucleotide sequence represented by GenBank ACC No.NM_007720.2.

CITATION LIST Non Patent Literature

-   [Non Patent Literature 1]-   Nat. Rev. Immunol., 2006, Vol. 6, No. 4, p. 295-307-   [Non Patent Literature 2]-   Eur. J. Immunol., 2010, Vol. 40, p. 3325-3335-   [Non Patent Literature 3]-   J. Clin. Oncol., 2006, Vol. 24, p. 5373-5380-   [Non Patent Literature 4]-   Nat. Med., 2004, Vol. 10, p. 942-949-   [Non Patent Literature 5]-   J. Clin. Oncol., 2007, Vol. 25, p. 2586-2593-   [Non Patent Literature 6]-   Cancer, 2006, Vol. 107, p. 2866-2872-   [Non Patent Literature 7]-   Eur. J. Cancer, 2008, Vol. 44, p. 1875-1882-   [Non Patent Literature 8]-   Cell. Mol. Immunol. 2011, Vol. 8, p. 59-66-   [Non Patent Literature 9]-   Cancer Res., 1999 July 1; Vol. 59, No. 13, p. 3128-33-   [Non Patent Literature 10]-   Cancer Res., 2010, Vol. 70, No. 7, p. 2665-74-   [Non Patent Literature 11]-   J. Immunol., 1996, Vol. 157, No. 7, p. 2759-63-   [Non Patent Literature 12]-   J. Biol. Chem., 1997, Vol. 272, No. 28, p. 17251-4

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to activate the immunity byinhibiting immunosuppression mediated by Treg cells or the like and toprovide a pharmaceutical composition for cancer treatment via thismechanism.

Solution to Problem

The present inventors have conducted diligent studies and consequentlycompleted the present invention by finding that tumor-infiltrating Tregcells and tumor-infiltrating macrophage cells specifically express CCR8,and the administration of an antibody against CCR8 decreases the cellcounts of the tumor-infiltrating Treg cells and the tumor-infiltratingmacrophage cells and inhibits tumor growth.

Specifically, the present invention relates to:

-   (1) a pharmaceutical composition for cancer treatment, comprising an    antibody against CCR8;-   (2) the pharmaceutical composition according to (1), wherein the    antibody against CCR8 is an antibody having ADCC activity;-   (3) the pharmaceutical composition according to (1) or (2), wherein    the antibody against CCR8 is a CCR8-neutralizing antibody;-   (4) the pharmaceutical composition according to any one of (1) to    (3), wherein the antibody against CCR8 has an effect of removing    tumor-infiltrating Treg cells;-   (5) the pharmaceutical composition according to any one of (1) to    (4), wherein the antibody against CCR8 has an effect of removing    tumor-infiltrating macrophage cells;-   (6) the pharmaceutical composition according to any one of (1) to    (5), wherein the cancer is breast cancer, colorectal cancer, kidney    cancer or sarcoma;-   (7) a medicament for cancer treatment, comprising a combination of    an antibody against CCR8 and an anti-PD-1 antibody or an anti-PD-L1    antibody;-   (8) a method for treating a cancer, comprising administering an    antibody against CCR8 according to any of one (1) to (5);-   (8-1) a method for treating a cancer, comprising administering an    antibody against CCR8;-   (8-2) the method according to (8-1), wherein the antibody against    CCR8 is an antibody having ADCC activity;-   (8-3) the method according to (8-1) or (8-2), wherein the antibody    against CCR8 is a CCR8-neutralizing antibody;-   (8-4) the method according to any one of (8-1) to (8-3), wherein the    antibody against CCR8 has an effect of removing tumor-infiltrating    Treg cells;-   (8-5) the method according to any one of (8-1) to (8-4), wherein the    antibody against CCR8 has an effect of removing tumor-infiltrating    macrophage cells;-   (8-6) the method according to any one of (8-1) to (8-5), wherein the    cancer is breast cancer, colorectal cancer, kidney cancer or    sarcoma;-   (8-7) the method according to any one of (8-1) to (8-6), further    administering an anti-PD-1 antibody or an anti-PD-L1 antibody;-   (9) the antibody against CCR8 according to any one of (1) to (5) for    treating a cancer;-   (9-1) an antibody against CCR8 for treating a cancer;-   (9-2) the antibody against CCR8 according to (9-1), wherein the    antibody against CCR8 is an antibody having ADCC activity;-   (9-3) the antibody against CCR8 according to (9-1) or (9-2), wherein    the antibody against CCR8 is a CCR8-neutralizing antibody;-   (9-4) the antibody against CCR8 according to any one of (9-1) to    (9-3), wherein the antibody against CCR8 has an effect of removing    tumor-infiltrating Treg cells;-   (9-5) the antibody against CCR8 according to any of one (9-1) to    (9-4), wherein the antibody against CCR8 has an effect of removing    tumor-infiltrating macrophage cells;

(9-6) the antibody against CCR8 according to any of one (9-1) to (9-5),wherein the cancer is breast cancer, colorectal cancer, kidney cancer orsarcoma; and

-   (9-7) a combination of an antibody against CCR8 according to any of    one (9-1) to (9-6) and an anti-PD-1 antibody or an anti-PD-L1    antibody for use in the treatment of a cancer.

Advantageous Effects of Invention

A pharmaceutical composition comprising the antibody of the presentinvention is pharmaceutically very useful for the treatment of cancers.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results of FACS analysis on kidney cancertumor-infiltrating CD3+CD4+ T cells. A CD25 molecule and a FoxP3molecule were each stained with an antibody and evaluated for theirexpression rates. CD25-expressing cells were found to also expressFoxP3.

FIG. 2 shows results of flow cytometry analysis on CD45RA and CD25expression intensity in peripheral blood mononuclear cells (hereinafter,referred to as PBMCs) of the same patient. CD3+CD4+ T cells werefractionated into 6 fractions (Fr1 to Fr6) as shown in the drawingaccording to CD45RA and CD25 expression levels, and cells in eachfraction were recovered using a sorter. The numeric values denote thecell abundance ratio (%) of each fraction. In this case, Treg fractionsare Fr1 and Fr2.

FIG. 3 shows results of flow cytometry analysis on CD45RA and CD25expression intensity in kidney cancer tumor-infiltrating cells.Tumor-infiltrating CD3+CD4+ T cells were fractionated into 4 fractions(Fr2 to Fr5) as shown in the drawing according to CD45RA and CD25expression levels, and cells in each fraction were recovered using asorter. The numeric values denote the cell abundance ratio (%) of eachfraction.

FIG. 4 shows results of conducting the RNA-Seq analysis of cells in eachof the fractions of FIGS. 2 and 3 and studying whether any of thesefractions would contain Treg cells on the basis of the mRNA expressionlevels of Treg-specific expressed genes FoxP3 and IKZF2. The ordinatedepicts a relative mRNA expression level after normalization. The strongintratumoral expression of both the genes was observed in Fr2 and Fr3.The strong expression of IL-2 or IFNγ, which is expressed in effectorcells, was observed in Fr4 and Fr5.

FIG. 5 shows results of analysis on a Treg-specific demethylation region(chrX, 49118000-49118500, hg19) at a FoxP3 gene locus in each fraction.Most of tumor-infiltrating CD3+CD4+ T cells in Fr2 and Fr3 fractionswere found to be Treg cells.

FIG. 6 shows results analyzing the mRNA expression level of CCR8 in eachfraction in the same way as in FIG. 4. Tumor-infiltrating Treg cellfractions Fr2 and Fr3 exhibited the strong expression of CCR8, whereinthe expression was rarely observed in Treg cells in peripheral bloodmononuclear cells (PBMCs).

FIG. 7 shows results of flow cytometry analysis on HEK293 cellsexpressing mouse CCR8. HEK293 cells were transfected with a pcDNA3.4expression vector having an insert of the mouse CCR8 gene anddrug-selected using G418. As for the degree of mouse CCR8 expression,the expression was confirmed with a PE-labeled anti-mouse CCR8 antibody.HEK293 cells transfected with a pcDNA3.4 vector and drug-selected in thesame way as above were used as a negative control. Almost all the cellswere found to express mouse CCR8.

FIG. 8 shows that an anti-mouse CCR8 antibody (SA214G2) has the abilityto activate a signaling pathway necessary for antibody-dependent cellmediated cytotoxicity (ADCC).

FIG. 9 shows that the anti-mouse CCR8 antibody (SA214G2) has ADCCactivity.

FIG. 10 shows that the anti-mouse CCR8 antibody (SA214G2) has activityof inhibiting intracellular calcium influx mediated by CCR8. An isotypecontrol antibody was used as a negative control.

FIG. 11 shows that the anti-mouse CCR8 antibody (SA214G2) does notrecognize CT26 cells. An isotype control antibody was used as a negativecontrol.

FIG. 12 shows results of administering a control antibody atpost-transplant day 3 to three BALB/c mice in which mouse colorectalcancer cell line CT26 cells were transplanted, excising tumors atpost-administration day 4 or 7, and analyzing the proportion of Tregcells present therein using a flow cytometer.

FIG. 13 shows results of analyzing the proportion of CCR8+ Treg cellsusing a flow cytometer in the same experiment as in FIG. 12.

FIG. 14 shows results of analyzing the proportion of CCR8-positive cellsin intratumoral CD11b+Gr1+CD206+M2 macrophage cells using a flowcytometer. In both cases, 40 to 50% cells were found to be CCR8-positiveM2 macrophage cells.

FIG. 15 shows the flow of an experiment of administering the anti-mouseCCR8 antibody (SA214G2) or an isotype control antibody atpost-transplant day 3 to BALB/c mice in which colorectal cancer cellline CT26 cells were transplanted, excising tumors at post-transplantday 7 or 10, and examining the abundance ratios of T lymphocytes andmacrophage cells present therein.

FIG. 16 shows the ratio of CD25+ FoxP3+ cells to CD45+CD4+ cells atpost-transplant day 7 (d7) or 10 (d10).

FIG. 17 shows the proportion of CD11b+F4/80+ macrophage cells atpost-transplant day 7 (d7).

FIG. 18 shows the abundance ratio of IA/IE-positive (IA/IE+) orIA/IE-negative cells (IA/IE−) at post-transplant day 7 (d7).

FIG. 19 shows the flow of an experiment of administering the anti-mouseCCR8 antibody (SA214G2) or an isotype control antibody (rat anti-KLH) ata single dose of 400 μg/mouse at post-transplant day 3 (d3) to BALB/cmice in which colorectal cancer cell line CT26 cells were transplanted,and measuring a tumor size every 3 to 4 days from post-transplant day 7(d7) up to day 21 (d21).

FIG. 20 shows results of measuring the solid tumor size of eachindividual after inoculation and calculating a tumor volume.

FIG. 21 shows the mean tumor volume of each mouse group at each point intime after inoculation. A standard deviation is also shown. Significancelevel *** denotes p<0.001, and significance level ** denotes p<0.01(t-test).

FIG. 22 2×10⁵ colorectal cancer cell line Colon26 cells wereintracutaneously transplanted to the back of each BALB/c mouse. Atpost-transplant day 3 (d3), the anti-mouse CCR8 antibody (SA214G2) or anisotype control antibody was administered at a single dose of 400μg/mouse. A tumor volume was measured every 3 to 4 days frompost-transplant day 3 (d3) up to day 18 (d18). The mean tumor volume ofeach group at each point in time after inoculation is shown.

FIG. 23 The plot shows the mean fluorescence intensity (MFI) of eachindividual in FACS analysis. The central horizontal lines depict themean MFI of 14 cases, and the vertical lines depict standard deviations.Significance level *** denotes P<0.001.

FIG. 24 shows an individual-based plot of the ratio of cells thatexhibited CCR8-positive signals (percent positivity) equal to or largerthan a background level obtained in an isotype control antibody, to CD3+CD4+ FoxP3+ T cells or CD3+ CD4+ FoxP3− T cells within the human kidneycancer tumors of 14 cases. The central horizontal lines depict the meanpercent positivity of the 14 cases, and the vertical lines depictstandard deviations.

FIG. 25 shows a Kaplan-Meier curve as to the survival probability ofeach group obtained by equally dividing clear cell renal cell carcinomapatients into 2 groups with high expression (High) and with lowexpression (Low) on the basis of the CCR8 mRNA expression levels ofintratumoral cells through the use of The Cancer Genome Atlas (TCGA)database. The ordinate depicts the survival probability, and theabscissa depicts the number of months. The numeric values denote thenumber of individuals in each group. The P value denotes a log-rank testvalue.

FIG. 26 shows results of analyzing prostate cancer patients in the sameway as in FIG. 25.

FIG. 27 shows results of analyzing bladder cancer patients in the sameway as in FIG. 25.

FIG. 28 shows that the anti-mouse CCR8 antibody recognizes neither MethAcells nor LM8 cells, as in FIG. 11. An isotype control antibody(Isotype) was used as a negative control.

FIG. 29 3×10⁵ osteosarcoma cell line LM8 cells were intracutaneouslytransplanted to the back of each C3H/He mouse. At post-transplant day 3(d3), the anti-mouse CCR8 antibody (SA214G2) or an isotype controlantibody (Control antibody) was administered at a single dose of 400μg/mouse. A tumor volume was measured every 3 to 4 days from 7 days upto 35 days after tumor inoculation. The mean tumor volume of each groupat each point in time after inoculation is shown. A standard deviationis also shown. Significance level *** denotes p<0.001, significancelevel ** denotes p<0.01, and significance level * denotes p<0.05(t-test).

FIG. 30 1×10⁵ MethA cells were intracutaneously transplanted to the backof each Balb/c mouse. At post-transplant day 3, the anti-mouse CCR8antibody (SA214G2) or an isotype control antibody (Control antibody) wasadministered at a single dose of 400 μg/mouse. A tumor volume wasmeasured every 3 to 4 days from 11 days up to 21 days after tumorinoculation. The mean tumor volume of each group at each point in timeafter inoculation is shown. Significance level * denotes p<0.05(t-test).

FIG. 31 1×10⁵ breast cancer cell line EMT6 cells were intracutaneouslytransplanted to the back of each Balb/c mouse. At 3 and 10 days aftertumor inoculation, the anti-mouse CCR8 antibody (SA214G2) or an isotypecontrol antibody was administered at 100 μg/mouse. A tumor volume wasmeasured every 3 to 4 days from 4 days up to 22 days after tumorinoculation. The mean tumor volume of each group at each point in timeafter inoculation is shown. Significance level *** denotes p<0.001, andsignificance level ** denotes p<0.01 (t-test).

FIG. 32 2×10⁵ colorectal cancer cell line Colon26 cells wereintracutaneously transplanted to the back of each BALB/c mouse. At 3 and10 days after tumor inoculation, an anti-isotype control antibody(Isotype antibody), the mouse CCR8 antibody (SA214G2) or an anti-PD-1antibody (RMP1-14) was administered at 400 μg/mouse. A tumor volume wasmeasured every 3 to 4 days from 3 days up to 24 days after tumorinoculation. The mean tumor volume of each group at each point in timeafter inoculation is shown.

FIG. 33 4×10⁵ mouse kidney cancer-derived cell line RAG cells wereintracutaneously transplanted to the back of each BALB/c mouse. 6 daysafter tumor inoculation, 100 μg (100 μL) of an isotype control antibody,the anti-mouse CCR8 antibody or an anti-mouse PD-1 antibody (Anti-PD-1antibody) was intraperitoneally administered thereto. A tumor volume wasmeasured every 3 to 4 days from 6 days up to 21 days after tumorinoculation. The mean tumor volume of each group at each point in timeafter inoculation is shown.

FIG. 34 2×10⁵ colorectal cancer cell line Colon26 cells wereintracutaneously transplanted to the back of each BALB/c mouse. At 3 and10 days after tumor inoculation, the anti-mouse CCR8 antibody (SA214G2)or an isotype control antibody (Control antibody) was administered at400 μg/mouse. 24 days after tumor inoculation, each organ was recoveredfrom the mice, and its weight was measured. The mean of 10 cases in eachgroup is shown.

FIG. 35 1×10⁵ mouse breast cancer cell line EMT6 cells wereintracutaneously transplanted to the back of each BALB/c mouse. Theanti-mouse CCR8 antibody was intravenously administered thereto at 3 and10 days after tumor inoculation, and an anti-mouse PD-1 antibody wasintravenously administered thereto at 8 and 13 days after tumorinoculation. An isotype control antibody was intravenously administeredto a control group at 3 and 10 days after tumor inoculation. A tumorvolume was measured every 3 to 4 days from 6 days up to 27 days afterinoculation. The mean tumor volume of each group at each point in timeafter inoculation is shown.

FIG. 36 shows the proportion of an individual bearing tumor larger than50 mm³ or smaller at each point in time after inoculation in each groupin the same experiment as in FIG. 35.

FIG. 37 4.5×10⁵ mouse kidney cancer-derived cell line RAG cells wereintracutaneously transplanted to the back of each BALB/c mouse. 8 and 15days after tumor inoculation, 100 μL of physiological saline, theanti-mouse CCR8 antibody or an anti-mouse PD-1 antibody, or theanti-mouse CCR8 antibody and the anti-mouse PD-1 antibody wasintravenously administered thereto. A tumor volume was measured every 3to 4 days from 8 days up to 33 days after tumor inoculation. The mediantumor volume of each group at each point in time after inoculation isshown.

FIG. 38 2×10⁵ CT26 cells were intracutaneously transplanted to the backof each wild-type mouse or homozygously CCR8 gene-deficient mouse ofBalb/c lineage (N=5). After inoculation, an isotype control antibody orthe anti-mouse CCR8 antibody was intravenously administered thereto. Atumor volume was measured every 3 to 4 days after tumor inoculation. Theleft diagram shows the mean tumor volume of the wild-type mice in eachgroup at each point in time after inoculation, and the right diagramshows the mean tumor volume of the homozygously CCR8 gene-deficient micein each group at each point in time after inoculation.

DESCRIPTION OF EMBODIMENTS

The pharmaceutical composition of the present invention comprises anantibody against CCR8.

The CCR8 of the present invention includes those derived from mice,rats, hamsters, guinea pigs, dogs, pigs, and primate mammals includingmonkeys and humans. Human CCR8 is preferred.

The antibody against CCR8 may be any of a human-derived antibody, amouse-derived antibody, a rat-derived antibody, a rabbit-derivedantibody and a goat-derived antibody as long as the antibody binds toCCR8. The antibody against CCR8 may be a polyclonal or monoclonalantibody thereof and may be any of a complete antibody, an antibodyfragment (e.g., a F(ab′)2, Fab′, Fab or Fv fragment), a chimericantibody, a humanized antibody and a complete human antibody. Ahuman-derived antibody, a humanized antibody or a complete humanantibody is preferred.

The antibody of the present invention can be produced according to anantibody or antiserum production method known in the art using afull-length protein or a partial protein of CCR8 as an antigen.Desirably, the antibody of the present invention binds to CCR8 expressedon cell surface. Therefore, the partial protein is desirably anextracellular region of CCR8. These antigens can be prepared by proteinexpression and purification methods known in the art.

Examples of the antigen, other than those described above, suitable forthe preparation of the antibody against CCR8 include cells forced toexpress CCR8 by an expression vector or the like, CCR8 expressionplasmid vectors, and CCR8 expression virus vectors (adenovirus vectors,etc.).

The polyclonal antibody can be produced by a method known in the art.The polyclonal antibody can be produced, for example, by immunizing anappropriate animal with an antigenic protein or a mixture thereof with acarrier protein, and harvesting a product containing an antibody againstthe antigenic protein from the immunized animal, followed by theseparation and purification of the antibody. Examples of the animal usedgenerally include mice, rats, sheep, goats, rabbits, and guinea pigs. Inorder to enhance the ability to produce antibodies, a complete Freund'sadjuvant or an incomplete Freund's adjuvant can be administered togetherwith the antigenic protein. In general, the administration is performeda total of approximately 3 to 10 times, usually once every approximately2 weeks. The polyclonal antibody can be harvested from the blood,ascitic fluid, or the like of the animal immunized by the methoddescribed above. A polyclonal antibody titer in antiserum can bemeasured by ELISA. The separation and purification of the polyclonalantibody can be performed according to an immunoglobulin separation andpurification method, for example, a purification method using an antigenbinding solid phase or an active adsorbent such as protein A or proteinG, a salting-out method, an alcohol precipitation method, an isoelectricprecipitation method, electrophoresis, an adsorption and desorptionmethod using an ion exchanger, an ultracentrifugation method, or a gelfiltration method.

The monoclonal antibody can be prepared by a known general productionmethod. Specifically, a mammal, preferably a mouse, a rat, a hamster, aguinea pig or a rabbit, is immune-sensitized with the antigen of thepresent invention, if necessary, together with a Freund's adjuvant, bysubcutaneous, intramuscular, intravenous, intra-footpad orintraperitoneal injection once to several times. Usually, immunizationwas performed once to 4 times every approximately 1 to 21 days frominitial immunization, and antibody-producing cells can be obtained fromthe immune-sensitized mammal approximately 1 to 10 days after the finalimmunization. The number of immunizations and the time interval can beappropriately changed according to the properties, etc. of the immunogenused.

Hybridomas secreting the monoclonal antibody can be prepared accordingto the method of Kohler and Milstein (Nature, 1975, vol. 256, p.495-497) and a method equivalent thereto. Specifically, the hybridomascan be prepared by the cell fusion of antibody-producing cells containedin the spleen, the lymph node, the bone marrow or the tonsil, etc.,preferably the spleen, obtained from a mammal immune-sensitized asmentioned above, with preferably mouse-, rat-, guinea pig-, hamster-,rabbit- or mammal (e.g. human)-derived, more preferably mouse-, rat- orhuman-derived myeloma cells lacking the ability to produce autologousantibodies.

In general, an established cell line obtained from mice, for example,P3-U1, NS-1, SP-2, 653, X63, or AP-1, can be used as the myeloma cellsfor use in the cell fusion.

A hybridoma clone producing the monoclonal antibody is screened for byculturing the hybridomas, for example, in a microtiter plate, measuringthe reactivity of a culture supernatant in a well where growth is seen,with the antigen of the present invention used in the mouse immunesensitization mentioned above by a measurement method such as RIA,ELISA, or FACS, and selecting a clone producing the monoclonal antibodythat exhibits specific binding to the antigen or hapten. Usually, amethod is further used which involves immobilizing the antigen on asolid phase, and detecting an antibody in a culture supernatant bindingthereto using a secondary antibody labeled with a radioactive material,a fluorescent material, an enzyme, or the like. In the case of usingantigen-expressing cells, the hybridoma culture supernatant is added tothe cells, and a fluorescently labeled secondary antibody can then bereacted therewith, followed by the measurement of fluorescence intensityof the cells using a fluorescent detection apparatus such as a flowcytometer to detect a monoclonal antibody capable of binding to theantigen of the present invention on the membranes of the cells.

The monoclonal antibody can be produced from the selected hybridoma byculturing the hybridoma in vitro or culturing the hybridoma in theascitic fluid or the like of a mouse, a rat, a guinea pig, a hamster ora rabbit, etc., preferably a mouse or a rat, more preferably a mouse,and isolating the monoclonal antibody from the obtained culturesupernatant or ascetic fluid of the mammal. For the in vitro culture,the hybridoma is grown, maintained and preserved according to variousconditions such as the characteristics of the cell type to be cultured,the purpose of a test and research and a culture method and can becultured using a known nutrient medium as used for producing monoclonalantibodies into a culture supernatant, or every nutrient medium inducedand prepared from a known basal medium.

Examples of the basal medium include low-calcium media such as Ham′ F12medium, MCDB153 medium and low-calcium MEM medium, and high-calciummedia such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium,ASF104 medium and RD medium. The basal medium can contain, for example,serum, hormone, cytokine and/or various inorganic or organic substances,according to a purpose.

The monoclonal antibody can be isolated and purified, for example, bysubjecting the culture supernatant or the ascetic fluid mentioned aboveto saturated ammonium sulfate, ion-exchange chromatography (DEAE orDE52, etc.), or affinity column chromatography using ananti-immunoglobulin column, a protein A column, or the like.

A recombinant antibody obtained by cloning an antibody gene fromantibody-producing cells, for example, hybridomas, integrating theantibody gene into an appropriate vector, and transfecting a host withthis vector, followed by production by use of a gene recombinationtechnique can be used as the antibody of the present invention (e.g.,Carl et al., THERAPEUTIC MONOCLONAL ANTIBODIES, published in 1990).

Specifically, mRNA encoding the variable region (V region) of theantibody is isolated from hybridomas producing the antibody of interestor immunocytes producing the antibody, for example, cells of sensitizedlymphocytes immortalized with an oncogene or the like. For the mRNAisolation, total RNA is prepared by a method known in the art, forexample, a guanidine ultracentrifugation method (Chirgwin, J. M. et al.,Biochemistry (1979) 18, 5294-5299), and the mRNA is prepared using mRNAPurification Kit (manufactured by Pharmacia Inc.) or the like.

cDNA of the antibody V region is synthesized from the obtained mRNAusing reverse transcriptase. The synthesis of the cDNA can be performedusing AMV Reverse Transcriptase First-strand cDNA Synthesis Kit or thelike. 5′-Ampli FINDER RACE Kit (manufactured by Clontech Laboratories,Inc) and PCR-based 5′-RACE (Frohman, M. A. et al., Proc. Natl. Acad.Sci. USA, 1988, Vol. 85, p. 8998, etc.) can be used for cDNA synthesisand amplification. The DNA fragment of interest is purified from theobtained PCR product and ligated with vector DNA. A recombinant vectoris further prepared therefrom. E. coli or the like is transfected withthe recombinant vector, and a colony is selected to prepare the desiredrecombinant vector. The nucleotide sequence of the DNA of interest isconfirmed by a method known in the art, for example, a deoxy method.

Provided that the DNA encoding the V region of the antibody of interestis successfully obtained, this DNA is linked to DNA encoding the desiredantibody constant region (C region) and the resultant is integrated intoan expression vector. Alternatively, the DNA encoding the V region ofthe antibody may be integrated into an expression vector containing theDNA of the antibody C region. In order to produce the antibody used inthe present invention, the antibody gene is integrated into anexpression vector such that the antibody gene is expressed under thecontrol of an expression control region, for example, enhancer/promoter.Next, host cells can be transformed with this expression vector toexpress the antibody.

For the expression of the antibody gene, DNA encoding the heavy chain (Hchain) and DNA encoding the light chain (L chain) of the antibody may beseparately integrated into expression vectors, with which a host isco-transformed, or the DNA encoding the H chain and the DNA encoding theL chain may be integrated into a single expression vector, with which ahost is transformed (see WO94/11523).

A so-called phage display technique (Nature Biotechnology 23, 1105(2005)) can also be used as a method, other than those described above,for preparing the antibody of the present invention. Specifically, forexample, an antibody gene library prepared by a method known in the artusing human or animal (e.g., rabbit, mouse, rat, or hamster) Blymphocytes as a material, or an antibody gene library completelysynthesized by selection and engineering from a human or animal germline sequence is displayed on, for example, bacteriophages, E. coli,yeast or animal cell surface, or liposomes. In this respect, examples ofthe form of the antibody to be displayed on the cell surface include IgGmolecules, IgM molecules, Fab fragments, and single-strand Fv (scFv)fragments.

The antibody fragment gene thus obtained can be recombined with acorresponding region of an IgG antibody gene by a method known in theart to obtain an antibody gene. Then, the gene thus obtained can beintegrated into an appropriate vector, with which a host is transfected,followed by the production of the antibody by use of a generecombination technique (e.g., Carl et al., THERAPEUTIC MONOCLONALANTIBODIES, published in 1990).

The antibody of the present invention includes antibodies artificiallyengineered for the purpose of, for example, reducing xenoantigenicityagainst humans, for example, chimeric antibodies, humanized antibodiesand complete human antibodies.

The antibody of the present invention may be a conjugated antibody inwhich the antibody is bound with any of various molecules such aspolyethylene glycol (PEG), radioactive substances, toxins, and sugarchains. Such a conjugated antibody can be obtained by chemicallymodifying the obtained antibody. The method for modifying the antibodyhas already been established in the art. The antibody according to thepresent invention also encompasses these conjugated antibodies.

The antibody of the present invention encompasses an antibody having aFc region bound with N-glycoside-linked sugar chains which are free froma fucose bound with N-acetylglucosamine at their reducing termini.Examples of the antibody having a Fc region bound withN-glycoside-linked sugar chains which are free from a fucose bound withN-acetylglucosamine at their reducing termini include antibodiesprepared using α1,6-fucosyltransferase gene-deficient CHO cells(International Publication Nos. WO 2005/035586 and WO 02/31140). Theantibody of the present invention having a Fc region bound withN-glycoside-linked sugar chains which are free from a fucose bound withN-acetylglucosamine at their reducing termini has high ADCC activity.

The antibody of the present invention may be fused at its N terminus orC terminus with an additional protein (Clinical Cancer Research, 2004,10, 1274-1281). The protein to be fused can be appropriately selected bythose skilled in the art.

The antibody fragment is a portion of the antibody of the presentinvention mentioned above and means a fragment having CCR8-specificbinding activity as in the antibody. Examples of the antibody fragmentcan specifically include Fab, F(ab′)2, Fab′, single-strand antibody(scFv), disulfide-stabilized antibody (dsFv), dimerized V regionfragment (diabody), and CDR-containing peptides (Expert Opinion onTherapeutic Patents, Vol. 6, No. 5, p. 441-456, 1996).

Alternatively, the antibody of the present invention may be a bispecificantibody which has two different antigenic determinants and binds todifferent antigens.

The ADCC (antibody-dependent cell mediated cytotoxicity) activity meansin vivo activity of damaging tumor cells or the like by activatingeffector cells via the binding of the Fc region of the antibody boundwith a cell surface antigen or the like on the tumor cells or the liketo a Fc receptor present on the effector cell surface. Examples of theeffector cells include natural killer cells and activated macrophages.

The antibody of the present invention is preferably an antibody havingADCC activity against cells expressing CCR8 because this antibody canremove Treg cells or macrophage cells. Whether or not the antibody ofthe present invention has such ADCC activity can be measured by, forexample, a method described in Examples mentioned later.

The antibody against CCR8 contained in the pharmaceutical composition ofthe present invention is preferably a CCR8-neutralizing antibody fromthe viewpoint of suppressing the intratumoral accumulation of Treg cellsor macrophage cells. The CCR8-neutralizing antibody means an antibodyhaving neutralizing activity against CCR8. Whether or not the antibodyof the present invention has neutralizing activity against CCR8 can bedetermined by measuring the presence or absence of suppression of thephysiological effect of CCL1 on CCR8. Examples thereof include, but arenot limited to, the measurement of the binding of CCL1 to CCR8, themigration of CCR8-expressing cells by CCL1, increase in intracellularCa⁺⁺ level by CCL1, and variation in the expression of a gene sensitiveto CCL1 stimulation. This can also be determined by a method describedin Examples mentioned later.

The antibody against CCR8 of the present invention preferably has aneffect of removing tumor-infiltrating Treg cells. Whether or not theantibody of the present invention has the effect of removingtumor-infiltrating Treg cells can be determined by, for example, amethod described in Examples mentioned later.

The antibody against CCR8 of the present invention preferably has aneffect of removing tumor-infiltrating macrophage cells. Whether or notthe antibody of the present invention has the effect of removingtumor-infiltrating macrophage cells can be determined by, for example, amethod described in Examples mentioned later.

The antibody of the present invention is useful as a pharmaceuticalcomposition. Thus, the pharmaceutical composition comprising theantibody of the present invention can be administered orally orparenterally and systemically or locally. For example, intravenousinjection such as infusion, intramuscular injection, intraperitonealinjection, subcutaneous injection, transnasal administration, orinhalation can be selected as parenteral administration.

The “cancer” for the “pharmaceutical composition for cancer treatment”of the present invention includes every solid cancer and blood cancer.Specifically, examples thereof include breast cancer, uterine corpuscancer, cervical cancer, ovary cancer, prostate cancer, lung cancer,stomach cancer (gastric adenocarcinoma), non-small cell lung cancer,spleen cancer, head and neck squamous cell carcinoma, esophageal cancer,bladder cancer, melanoma, colorectal cancer, kidney cancer, non-Hodgkinlymphoma, urothelial cancer, sarcoma, blood cell carcinoma (leukemia,lymphoma etc.), bile duct carcinoma, gallbladder carcinoma, thyroidcarcinoma, prostate cancer, testicular carcinoma, thymic carcinoma, andhepatocarcinoma. Preferably, examples thereof include breast cancer,uterine corpus cancer, ovary cancer, lung cancer, colorectal cancer,kidney cancer and sarcoma, and more preferably, examples thereof includebreast cancer, colorectal cancer, kidney cancer, and sarcoma.

The “cancer” for the “pharmaceutical composition for cancer treatment”of the present invention is preferably a cancer expressing atumor-specific antigen.

The “cancer” described in the present specification means not onlyepithelial malignant tumors such as ovary cancer and stomach cancer butnon-epithelial malignant tumors including hematopoietic cancers such aschronic lymphocytic leukemia and Hodgkin lymphoma. In the presentspecification, terms such as “cancer”, “carcinoma”, “tumor”, and“neoplasm” can be used interchangeably with each other withoutdifferentiating thereamong.

The antibody against CCR8 of the present invention may be administeredas a concomitant drug in combination with an additional drug in order to

-   (1) complement and/or potentiate the therapeutic effect of the    pharmaceutical composition of the present invention,-   (2) improve the pharmacokinetics and absorption of the    pharmaceutical composition of the present invention, and reduce the    dose thereof, and/or-   (3) reduce the adverse reaction of the pharmaceutical composition of    the present invention.

The concomitant drug of the antibody against CCR8 of the presentinvention and an additional drug may be administered in the form of acombination drug containing both the ingredients in one preparation ormay be administered in the form of separate preparations. Thisadministration as separate preparations includes concurrentadministration and staggered administration. For the staggeredadministration, the antibody of the present invention may beadministered first, and the additional drug may be administered later,or the additional drug may be administered first, and the compound ofthe present invention may be administered later. Their respectiveadministration methods may be the same or different.

Examples of the additional drug that may be used in combination with theantibody against CCR8 of the present invention include anti-PD-1antibodies, anti-PD-L1 antibodies and anti-CTLA-4 antibodies. Ananti-PD-1 antibody or an anti-PD-L1 antibody is preferred, and ananti-PD-1 antibody is more preferred.

In the present invention, examples of the anti-PD-1 antibody includenivolumab and pembrolizumab.

In the present invention, examples of the anti-PD-L1 antibody includeatezolizumab, avelumab, and durvalumab.

In the present invention, examples of the anti-CTLA-4 antibody includeipilimumab.

The patient intended by the pharmaceutical composition of the presentinvention is expected to be a cancer patient or a patient suspected ofhaving a cancer. The effective dose is selected from the range of 0.01mg to 100 mg per kg of body weight per dose. Alternatively, the dose canbe selected from 5 to 5000 mg, preferably 10 to 500 mg, per patient.However, the pharmaceutical composition comprising the antibody of thepresent invention or an antibody fragment thereof is not limited bythese doses. Also, the dosing period can be appropriately selectedaccording to the age and symptoms of the patient. The pharmaceuticalcomposition of the present invention may further contain apharmaceutically acceptable carrier or additive depending on anadministration route. Examples of such a carrier and additive includewater, pharmaceutically acceptable organic solvents, collagen, polyvinylalcohol, polyvinylpyrrolidone, sodium alginate, water-soluble dextran,pectin, methylcellulose, ethylcellulose, casein, diglycerin, propyleneglycol, polyethylene glycol, Vaseline, human serum albumin (HSA),mannitol, sorbitol, lactose, and surfactants acceptable aspharmaceutical additives. The additive used is selected appropriately orin combination from among those described above according to a dosageform, though the additive is not limited thereto.

Hereinafter, the present invention will be specifically described withreference to Examples. However, the present invention is not limited byExamples given below. Methods described in Molecular Cloning: ALaboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory) were usedas gene manipulation approaches unless otherwise specified.

Example 1 Extraction and Analysis of Kidney Cancer Tumor-InfiltratingCells and PBMCs

The following analysis was conducted using a portion of primary tumortissues excised by surgical treatment from clear cell renal cellcarcinoma (ccRCC) patients (3 cases) who were not preoperatively treatedwith an anticancer agent, radiation, or the like. After tumor weightmeasurement, tumor masses were cut into 2 mm square with scissors, andtumor tissue homogenates were prepared using Tumor Dissociation Kit,human (130-095-929, Miltenyi Biotec) and gentleMACS™ Dissociator(Miltenyi Biotec, 130-093-235) according to the protocol attached to thekit. The homogenates were passed through a 70 um cell strainer andsubjected to hemolysis treatment, followed by the removal of debris anddead cells in a solution of 30% Percoll in PBS to obtain tumor tissuesingle cells.

Peripheral blood mononuclear cells (PBMCs) of the same patient wereseparated from peripheral blood by the density gradient centrifugationmethod using Ficoll-Paque PLUS (GE Healthcare Japan Corp.). After cellcount measurement, the separated intratumoral cells and PBMCs weretreated with Human TruStain FcX™ (BioLegend, Inc., 422-301) and ZombieNIR™ Fixable Viability kit (BioLegend, Inc., 423105) according to theattached protocols and stained 30 minutes in ice. Then, the cells werewashed once with 2% FCS/HEPES/HBSS and then stained with the followinglabeling antibodies according to the protocols attached to the labelingantibodies.

The cell surface of tumor-infiltrating cells was stained throughreaction for 30 minutes in ice using an anti-CD3 antibody (BioLegend,Inc., Clone UCHT1), an anti-CD4 antibody (BioLegend, Inc., Clone OKT4),and an anti-CD25 antibody (BioLegend, Inc., Clone BC96). The cells werewashed twice with 2% FCS/HEPES/HBSS and then fixed andmembrane-permeabilized using Foxp3/Transcription Factor Staining BufferSet (eBioscience, Inc., 00-5523-00) according to the protocol attachedto the kit. FoxP3 was further stained using a PE-labeled anti-FoxP3antibody (eBioscience, Inc., Clone PCH010). The cells were washed oncewith a washing solution attached to the kit and then analyzed by flowcytometry (BD Biosciences, BD LSRFortessa). Almost all the CD4+CD25+ Tcells within the ccRCC tumors were confirmed to express FoxP3, a markerof Treg cells (FIG. 1).

Subsequently, the tumor-infiltrating cells and the PBMCs described abovewere stained with an anti-CD3 antibody, an anti-CD4 antibody, ananti-CD45RA antibody (BD Biosciences, Clone HI100) and an anti-CD25antibody. CD3+CD4+ T cells were two-dimensionally developed on the basisof CD45RA and CD25 expression levels. The results about the PBMCs areshown in FIG. 2, and the results about the tumor-infiltrating cells areshown in FIG. 3. The tumor-infiltrating cells were fractionated into 4fractions of strongly positive cells (Fr2), weakly positive cells (Fr3),and negative cells (Fr4 and Fr5) as shown in FIG. 1C withCD3+CD4+CD45RA- and CD25 expression intensity as an index using a cellsorter (FACSAria II), and cells contained in each fraction wererecovered. The PBMCs were also two-dimensionally developed, as in thetumor-infiltrating cells, and fractionated into Fr1 to Fr6 as shown inFIG. 2 with CD45RA and CD25 expression intensity as an index, and cellscontained in each fraction were recovered.

Example 2

Separation of RNA from Fractionated Cells and cDNA Sequence Analysis

The cells separated and recovered from each fraction were lysed in RLTbuffer (Qiagen N.V.), and total RNA was extracted using AgencourtRNAClean XP (Beckman Coulter, Inc.). The recovered RNA was prepared intocDNA using SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (ClontechLaboratories, Inc.), and a library was prepared using KAPA Hyper PrepKit for illumina (Kapa Biosystems, Inc.). For the cDNA synthesis and thelibrary preparation, quality control was constantly performed usingAgilent 2100 Bioanalyzer (Agilent Technologies, Inc.) to confirm thatthese procedures were free from problems. The finished cDNA library wastitrated using a KAPA library Quantification kit Illumina Platforms(Kapa Biosystems, Inc.). Then, DNA sequencing was performed by pairedend reads using Hiseq 4000 (Illumina, Inc.) to obtain 20,000,000 readsor more of 100-base pair sequence data per sample (Fastq file).

The raw data (Fastq file) was analyzed by FastQC, and adaptor sequencesand repeat sequences were removed using CutAdapt. Pairs of each pairedend read were matched using cmpfastq_pe program. hg38 was used as areference sequence in genome mapping, and the reads were mapped onto thegenome at default setting using TOPHAT2 program having Bowtie 2. Themapped reads were sequence-sorted using SAMtools program and countedusing HTSEQ program. The count data was normalized using Deseq 2program. Among the obtained fractions, a fraction containing Treg cellswas confirmed by the following method.

Treg cells are known to constitutively express FoxP3 and Ikzf2 genes asmarker genes and to rarely secrete IFNγ or IL2 even when activated bystimulation. Whether or not to contain Treg cells may be confirmed tosome extent by examining the expression levels of these genes. As aresult of examining the expression levels of these genes as to eachfraction of the tumor-infiltrating cells and the PBMCs on the basis ofthe RNA-Seq data described above, Ikzf2 and FoxP3 were found to bespecifically expressed in Fr2 and Fr3 of the tumor-infiltrating cellsand Fr2 of the PBMCs and rarely expressed in the other fractions (FIG.4). Also, IFNγ (IFN-gamma) and IL2 were found to be specificallyexpressed in Fr4 and Fr5 of the tumor-infiltrating cells and Fr4 and Fr5of the PBMC cells and not expressed in the other fractions (FIG. 4). Inconclusion, the Treg cells were found to be contained in Fr2 and Fr3 ofthe tumor-infiltrating cells and Fr2 of the PBMCs and not contained inthe other fractions.

Example 3 Measurement of Demethylation Rate of FoxP3 Region

The demethylation rate of a FoxP3 region serves as an index foraccurately determining the proportion of Treg cells. Therefore, thecells in Fr2 to Fr5 of the kidney cancer tumor-infiltrating cellsobtained as described above were studied for the demethylation rate ofthe FoxP3 region. A region demethylated in a Treg cell-specific mannerresides (chrX, 49118000-49118500, hg19) in a particular CpG regionwithin the first intron of the FoxP3 gene. The cells contained in eachfraction of the tumor-infiltrating cells may be analyzed for thedemethylation of this region to verify whether the fraction obtainedthis time consists of only Treg cells or other cells also coexisttherewith.

Each fraction (Fr2, Fr3, Fr4, and Fr5) of the tumor-infiltrating CD4+ Tcells was recovered, and genome DNA was recovered by use of the phenolextraction method. The genome DNA was treated with bisulfite usingMethylEasy Xceed kit (Human Genetic Signatures), and the FOXP3 intron 1region (chrX, 49118000-49118500, hg19), a Treg cell-specificdemethylation region, was subjected to amplicon PCR. DNA methylation wasdetected using a prepared methylated DNA-specific FAM fluorescent probeand demethylation-specific VIC fluorescent probe and QuantStudio 3Ddigital PCR system (Applied Biosystems, Inc.). After the amplicon PCR,the numbers of light emissions from the FAM and VIC fluorescent probeswere counted, and the DNA methylation rate was calculated from the ratiobetween these numbers of fluorescence emissions and used as themethylation rate of each fraction (Fr2 to Fr5).

As a result, 95% or more CpG sequences within the FOXP3 intron 1 region(chrX, 49118000-49118500) were demethylated in the cells contained inFr2 and Fr3 of the tumor-infiltrating cells, whereas the demethylationrates of Fr4 and Fr5 were 50% or less. In conclusion, almost all thecells contained in Fr2 and Fr3 were found to be Treg cells (FIG. 5).

Example 4 Identification of CCR8

In order to identify a gene of one group specifically expressed in theTreg cells (Fr2 of the tumor-infiltrating cells), hierarchicalclustering analysis was conducted on the gene expression data on thePBMC-derived CD4+ T cell fraction of the same patient as in eachtumor-derived CD4+ T cell fraction. CCR8 was identified as a gene thatwas expressed in Fr2 of the Treg cells and rarely expressed intumor-derived Fr5 and Fr4 and Fr5 of the PBMCs (FIG. 6).

Example 5 Preparation of Cells Forced to Express Mouse CCR8

Full-length ORF of mouse CCR8 (hereinafter, also referred to as mCCR8)was inserted to an expression vector (pcDNA3.4) to constructpcDNA3.4-mCCR8 plasmid. The nucleotide sequence was changed to havecodons with high usage frequency in mammals without changing the aminoacids. HEK293 cells were transfected with pcDNA3.4 or the pcDNA3.4-mCCR8expression plasmid using Lipofectamine 3000 and drug-selected at ageneticin (G418) concentration of 1 mg/ml for 2 weeks.

Surviving cells were dissociated with trypsin and washed with DMEM/10%FCS medium. Then, a PE-labeled anti-mCCR8 antibody (clone SA214G2)diluted 1/200 was added thereto and reacted on ice for 30 minutes. Then,the cells were washed once with DMEM/10% FCS to label mCCR8 expressed onthe cell surface. A cell population expressing mCCR8 was enriched bysorting using a cell sorter (FACSAria II). The positive cell populationwas cultured at 37° C. for 2 weeks in a CO2 incubator in the presence ofDMEM/10% FCS (medium containing 1 mg/ml G418). For the cells transformedwith pcDNA3.4, only drug selection was performed, and sorting was notperformed. In order to confirm expression, both the cells were stainedwith a commercially available anti-PE-labeled anti-mouse CCR8 antibody(clone SA214G2) and analyzed using a flow cytometer (FACSAria II). Theresults are shown (FIG. 7). The expression of mCCR8 was observed in 99%or more of the cells transformed with pcDNA3.4-mCCR8 compared with thecells transformed with pcDNA3.4.

Example 6 Study on Ability of Anti-Mouse CCR8 Antibody (SA214G2) toStimulate FcγR

An anti-mouse CCR8 antibody (clone SA214G2, purchased from BioLegend,Inc.) was evaluated for the ability to stimulate FcgR, necessary for itsADCC activity, using mFcγRIV ADCC Reporter Bioassays Core kit (PromegaCorp.). This kit indicates the activation of FcγR on effector cells bythe expression level of luciferase gene linked downstream of NFATpromoter in the cells. The activation of FcγR signals can be quantifiedby quantifying this expression level.

Hereinafter, the procedures will be briefly described. 1×10⁵ cells/wellof mCCR8-expressing HEK293 target cells (target cells) dissociated withtrypsin were mixed with FcγR-expressing effector cells attached to thekit at a ratio of 1:1.5 in a 96-well plate. Immediately after the cellmixing, the antibody against mCCR8 was added thereto. The concentrationwas set to 33 ug/ml to 0.033 ug/ml as shown in FIG. 8 (N=2). Only theeffector cells were used as a negative control. 14 hours after theantibody addition, the cells were recovered, and the luciferase activitywas measured (FIG. 8). A mean of N=2 is shown.

As a result, the luciferase activity was not observed at any of theantibody concentrations for the negative control, whereas antibodyconcentration-dependent activity was observed in the target celladdition group. The ordinate depicts a relative value of luminescenceintensity. As seen from FIG. 8, the largest activity value wasapproximately 6000 relative light units (R.L.U), and the EC50 value(approximately 3500 R.L.U) was approximately 0.1 μg/ml (lines in thedrawing). These results demonstrated that the anti-mouse CCR8 antibody(SA214G2) can activate FcγRIV.

Example 7 Measurement of ADCC Activity

The anti-mCCR8 antibody (SA214G2) was evaluated for its cytotoxicactivity using the stably mCCR8-expressing HEK293 cells prepared inExample 5.

The spleen of a C57BL/6 mouse was separated, and spleen cells wererecovered through a cell strainer. The cells were washed and thenreacted with a biotinylated anti-CD49b (clone DX5) antibody at 4° C. for30 minutes. After washing, NK cells were purified using streptavidinmicrobeads (Miltenyi Biotec) and used as effector cells. The HEK293cells expressing mouse CCR8 were stained with Cell Trace Violet (CTV)(Thermo Fisher Scientific Inc., C34557) at a final concentration of 2.5uM and used as target cells. These cells were mixed at a ratio ofeffector cells:target cells=5:1 (effector cell count: 2.5×10⁵ cells) ina 96-well plate (200 μL/well). The anti-mouse CCR8 antibody or anisotype control antibody (rat IgG2b, clone RTK4530) was added thereto ata final concentration of 1 μg/ml, followed by overnight culture in a CO2incubator of 37° C. Then, PE-labeled annexin V (Annexin V-PE, Medical &Biological Laboratories Co., Ltd. (MBL), 4696-100) diluted 1/100 wasadded according to the attached protocol, and the cells were stained at37° C. for 30 minutes and then washed once. The proportion of annexinV-positive apoptotic cells in the CTV-stained target cells was analyzedusing a flow cytometer. The test was carried out in triplicate (N=3),and a mean and a standard deviation thereof are shown. A typical exampleof two similar experiments is shown (FIG. 9). The addition of theanti-mouse CCR8 antibody compared with the isotype control antibodysignificantly increased the proportion of annexin V-positive cells inthe target cells by approximately 6 times. In conclusion, the anti-mouseCCR8 antibody (SA214G2) was found to have ADCC activity.

Example 8 Measurement of Neutralizing Activity Against CCR8

The anti-mouse CCR8 antibody (SA214G2) was evaluated for itsneutralizing activity against CCR8 with intracellular calcium influxmediated by mouse CCL1 (ligand of mouse CCR8) as an index using HEK293cells stably expressing mouse CCR8.

The following reagents were used in calcium measurement. HEPES (WakoPure Chemical Industries, Ltd., CAS. NO. 7365-45-9) HBSS(+) withoutPhenol Red (Wako Pure Chemical Industries, Ltd.) Fluo 3-AM (cat F023,Dojindo Laboratories) Probenecid (CAS-No: 57-66-9, Nacalai Tesque, Inc.)Pluronic F127 (P3000MP; Life Technologies Corp.) 10 mM HEPES/HBSS/0.1%BSA Buffer (HEPES (final concentration: 10 mM) and BSA (finalconcentration: 0.1%) were added to HBSS)

Fluo 3-AM and Pluronic F127 were dissolved at final concentrations of 4μmol/L and 0.04%, respectively, in 10 mM HEPES/HBSS Buffer. The cellswere suspended in this solution and incubated at 37° C. for 1 hour sothat Fluo 3-AM was taken up by the cells. Then, the cells were washedthree times with 10 mM HEPES/HBSS/0.1% BSA solution and suspended at acell concentration of 2×10⁵ cells/ml in 10 mM HEPES/HBSS/0.1% BSAsolution containing 1.25 uM probenecid. Then, the cells were incubatedat 37° C. for 10 minutes in a CO2 incubator. The anti-mCCR8 antibody(SA214G2) or an isotype control antibody (Clone LTF-2, Bio X Cell) wasfurther added thereto at a concentration of 5 μg/ml. The cells werefurther incubated at 37° C. for 20 minutes.

2 mL of the solution of the cells was placed in a quartz glass cuvetteand loaded in a spectrophotometer HITACHI F7000 with the temperature ofa measurement room preset to 35° C. The measurement conditions were asdescribed below.

Excitation wavelength: 508.0 nm, fluorescence (measurement) wavelength:527.0 nm, excitation-side slit: 5 nm, fluorescence-side slit: 5 nm,photomultiplier voltage: 950 V, response: 0.5 s

The cells were incubated with stirring using a stirrer for approximately30 seconds until the fluorescence wavelength was stabilized. When thewavelength was stabilized, mouse CCL1 was added thereto at a finalconcentration of 50 nM (4 μL) to start measurement. As a result of themeasurement, the administration of the anti-mCCR8 antibody in advancewas found to almost completely suppress intracellular calcium influxmediated by mCCL1 (FIG. 10). Such suppression was not observed by theaddition of the control antibody. The gaps in the graphs were derivedfrom the opening and closing of the cover of the instrument in order toadminister the agonist to the cells. In conclusion, the anti-mCCR8antibody (SA214G2) was found to have neutralizing activity against mouseCCR8.

Example 9

Confirmation of Expression of mCCR8 in CT26

CT26 cells were cultured in a 6-well dish, and the culture solution wasremoved when the cells became approximately 50% confluent. 5 ml of 10 mMEDTA/PBS was added thereto, and the cells were incubated at 37° C. for 5minutes. As a result, almost all the cells were dissociated, suspendedusing a pipette and were thereby able to be separated into almost singlecells. The cells were washed twice with D-MEM/10% FCS, suspended inD-MEM/10% FCS, and stained in ice with LIVE/DEAD® Fixable Near-IR DeadCell Stain Kit (Thermo Fisher Scientific Inc., L34975) and anAPC-labeled anti-mCCR8 (SA214G2) or APC-labeled isotype controlantibody. 1 hour later, the cells were washed three times with D-MEM/10%FCS and analyzed for a mCCR8 expression rate using a flow cytometer(FACSCanto II). A background was set using the isotype control antibody,and the proportion of positive cells (P6) equal to or larger than thebackground level and median APC fluorescence were calculated (FIG. 11).As a result, no difference in median APC fluorescence intensity wasobserved, and the positive cells were rarely observed (0.2%). Inconclusion, the CT26 cells were not recognized by the anti-mCCR8antibody, and the CT26 cells were confirmed to not express mCCR8.

Example 10 Confirmation of CCR8 Expression in Tumor-Infiltrating CellsUsing Colorectal Cancer Cell Line CT26 Cells

3×10⁵ CT26 cells (50 μL) were intracutaneously transplanted to the backof each Balb/c mouse (7 w, female) (N=3). At post-transplant day 3, 400μg of a rat anti-KLH (keyhole limpet hemocyanin, Clone LTF-2) antibody(IgG2b) was intraperitoneally administered thereto. Atpost-administration days 4 (4d) and 7 (7d), tumors were recovered fromthe 3 individuals (N=3). The tumor masses of the CT26 cells were choppedwith scissors, and tumor-infiltrating cells were prepared using acommercially available kits (Tumor Dissociation Kit, mouse, MiltenyiBiotec and gentleMACS™ Dissociator, Miltenyi Biotec, cat. 130-095-929)according to the protocols attached to the kits.

The prepared cells were passed through a 70 um cell strainer and thenwashed twice with 10 mM HEPES/HBSS/2% FBS. Then, the cells were treatedwith an erythrocyte lysis solution (Miltenyi Biotec) for 5 minutes forthe removal of erythrocytes and further washed twice with 2% FCS (fetalcalf serum)/10 mM HEPES/HBSS buffer. The tumor-infiltrating cells weredivided into two parts, one of which was used in the identification ofTreg cells and the other of which was used in the identification ofmyeloid (macrophage) cells. The cells were stained using the followingmethod and antibodies. The antibodies, staining reagents, and assaybuffers used were as described below.

The following antibodies were used.

(Antibody set for Treg cell confirmation)PE anti-mouse/rat FoxP3 (clone FJK-16s), eBioscience, Inc.Anti-mouse CD4 PerCP/Cy5.5 (clone RM4-5), eBioscience, Inc.Anti-mouse CD8a FITC (clone 5H10-1), BioLegend, Inc.Bv421 anti-mouse CD25 (clone PC61), BioLegend, Inc.Bv510 anti-mouse CD45 (clone 30-F11), BioLegend, Inc.AF647 Anti-mouse CCR8 (clone SA214G2), BioLegend, Inc.AF647 Isotype Control (clone RTK4530), BioLegend, Inc. (CCR8-negativecontrol)(Antibody set for myeloid and macrophage cell confirmation)AF647 Anti-mouse CCR8 (clone SA214G2), BioLegend, Inc.AF647 Isotype Control (clone RTK4530), BioLegend, Inc. (CCR8-negativecontrol)Bv510 anti-mouse CD45 (clone 30-F11), BioLegend, Inc.FITC anti-mouse Gr-1 (clone RB6-8C5), BioLegend, Inc.Bv421 anti-mouse F4/80 (clone BM8), BioLegend, Inc.PECy7 anti-mouse CD11b (clone M1/70), BioLegend, Inc.PerCP/Cy5.5 Anti-mouse MHC class II IA/IE (clone M5/114.15.2),BioLegend, Inc.PE anti-mouse CD206 (clone C068C2), BioLegend, Inc.(Other reagents used)

Zombie NIR Fixable Viability Kit (cat no. 423106), BioLegend, Inc.

BD Pharmingen Transcription Factor buffer Set (cat no. 562574)

BD Pharmingen Lysing Buffer (cat no. 555899) HBSS(−), Wako Pure ChemicalIndustries, Ltd., 084-08345 FCS (HyClone Laboratories Inc., cat no.5H30070.03)

The staining method was as follows: the infiltrating cells were stainedin ice for 30 minutes using a reagent of Zombie NIR Fixable ViabilityKit. The cells were washed once with 2% FCS/10 mM HEPES/HBSS. Then,Treg- and CCR8-positive cells were stained with Bv510-labeled anti-CD45,PerCP/Cy5.5-labeled anti-mouse CD4, FITC-labeled anti-mouse CD8,Bv421-labeled anti-mouse CD25, and AF647-labeled anti-mouse CCR8antibody (or AF647-labeled isotype control antibody). Monocytic cellswere stained with Bv510-labeled anti-CD45, FITC anti-mouse Gr-1, PECy7anti-mouse CD11b, Bv421 anti-mouse F4/80, PerCP/Cy5.5-labeled MHC class2 (IA/IE) antibody, and PE-labeled anti-mouse CD206 antibody.

The staining was carried out in ice for 30 minutes. The cells werewashed twice with 2% FCS/HEPES/HBSS and then fixed using a commerciallyavailable kit (FoxP3 staining kit, eBioscience, Inc.) according to theattached protocol, and intracellular FoxP3 was stained using aPE-labeled anti-FoxP3 antibody. The cells were washed with a bufferattached to the kit and then analyzed using a flow cytometer.

CD45+CD4+ T cells were analyzed. A negative cell region in the CD45+CD4+T cells was determined by staining with an isotype control antibody, andcells positive to both anti-mouse CD25 and anti-mouse FoxP3 antibodieswere used as Treg cells to calculate the frequency of presence 4 daysafter administration (7 days after inoculation) and 7 days afteradministration (10 days after inoculation). As a result, approximately23% (4d) and approximately 30% (7d) of the CD45+CD4+ cells within themouse tumors were CD25+ FoxP3+ cells (FIG. 12).

Next, CCR8 expression in the CD45+CD4+CD25+ FoxP3+ T cells was analyzed.A negative cell region in the CD45+CD4+CD25+ FoxP3+ cells was determinedby staining with an isotype control antibody, and cells positive to ananti-mouse CCR8 antibody were used as CCR8+ Treg cells to calculate thefrequency of presence 4 days after administration (7 days afterinoculation) and 7 days after administration (10 days after inoculation)(FIG. 13). As a result, approximately 50% (4d) and approximately 67%(7d) of the CD45+CD4+CD25+ FoxP3+ T cells within the mouse tumors wereCCR8+ cells (FIG. 13).

As for myeloid cells, the myeloid population was gated on CD45+ cellsand FSC/SSC using a flow cytometer and analyzed for the proportion ofCCR8+ cells in CD11b+Gr1+CD206+ cells. As a result, 40 to 50% cells both7 days after inoculation (4 days after administration) and 10 days afterinoculation (7 days after administration) were found to be CCR8-positive(FIG. 14). Also, the CCR8 expression rate in CD45+CD11b+F4/80+ cells(N=3) as a macrophage cell population different therefrom was measuredin the same way as above. As a result, 45.3% (standard deviation: ±8.2%)of the cells were confirmed to express CCR8 at post-transplant day 10(7d). From these results, at least CD4+CD25+ FoxP3+ T cells andCD11b+Gr1+CD206+ macrophages (called M2 macrophages) astumor-infiltrating cells were found to express CCR8.

Example 11 Study on Effect of Removing Tumor-Infiltrating Treg Cells orTumor-Infiltrating Macrophage Cells by Anti-mCCR8 AntibodyAdministration

3×10⁵ CT26 cells (50 uL) were intracutaneously transplanted to the backof each Balb/c mouse (7 w, female). 3 days after inoculation, 400 μg(liquid volume: 400 μL) of a rat anti-mouse CD198 (CCR8) antibody (cloneSA214G2, BioLegend, Inc.) or an isotype control antibody (Clone LTF-2)was administered into the tail vein (each group N=3). 7 days after tumorinoculation (4 days after antibody administration) and 10 days aftertumor inoculation (7 days after antibody administration), tumors wererecovered, and tumor-infiltrating cells were prepared and analyzed (FIG.15).

Tumor-infiltrating Treg cells were recovered in the same way as inExample 10. The antibodies used were the same as in Example 10.

First, the infiltrating cells were stained in ice for 30 minutes usingZombie NIR Fixable Viability Kit. The cells were washed once with 2%FCS/10 mM HEPES/HBSS and then stained with Bv510-labeled anti-CD45,PerCP/Cy5.5-labeled anti-mouse CD4, FITC-labeled anti-mouse CD8antibody, Bv421-labeled anti-mouse CD25, and AF647-labeled anti-mouseCCR8 antibody (or AF647-labeled isotype control antibody). The stainingwas carried out in ice for 30 minutes. The cells were washed twice with2% FCS/HEPES/HBSS and then fixed using a commercially available kit(FoxP3 staining kit, eBioscience, Inc.) according to the attachedprotocol, and intracellular FoxP3 was stained using a PE-labeledanti-FoxP3 antibody. The cells were washed with a buffer attached to thekit and then analyzed using a flow cytometer.

CD45+CD4+ FoxP3+CD25+ cells were used as mouse Treg cells. A negativecell region in the Treg cells was determined by staining with anAF647-labeled isotype control antibody, and cells positive to anAF647-labeled anti-mouse CCR8 antibody compared with the control wereused as CCR8-positive cells to calculate the frequency thereof.

As a result, as shown in FIG. 16, the percent positivity ofCD45+CD4+CD25+ FoxP3+ T cells (Treg cells) in the mice given theanti-mouse CCR8 (SA214G2) antibody was approximately 80% 7 days aftertumor inoculation (4 days after antibody administration) andapproximately 40% 10 days after tumor inoculation (7 days after antibodyadministration 7) (FIG. 16) when the proportion of intratumoralCD45+CD4+CD25+ FoxP3+ T cells (Treg cells) in the mice given the isotypeantibody was defined as 100% (10 days after tumor inoculation).Significance level ** was P<0.01 (t test). These results showed thatapproximately 60% of the tumor-infiltrating Treg cells were removed bythe anti-CCR8 antibody 7 days after anti-CCR8 antibody administration.

In the same way as above, tumor-infiltrating cells were separated fromtumors at post-transplant day 7 (d7), and among CD45+ cells, a myeloidpopulation was gated on FSC/SSC (referred to as FSC/SSC+), followed bythe analysis of CD11b+F4/80+ cells in the cells. F4/80 (Ly719) is amarker of mouse mature macrophages and monocytes. As shown in FIG. 17,the abundance ratio of CD11b+F4/80+ cells was decreased in theanti-mCCR8 antibody administration group (N=3) compared with the isotypecontrol (N=3) (t test; P=0.062). The graph shows the abundance ratio ofF4/80+ cells in a CD45+ FSC/SSC+ mononuclear cell population.

The abundance ratio of IA/IE-positive or class 2 (IA/IE)-negative cellsin the F4/80+ cells shown in FIG. 17 is further shown as to MHC (tumorhistocompatibility antigen) class 2 molecules. As shown in FIG. 18, inthe anti-mCCR8 antibody administration group (N=3) compared with theisotype control (N=3), the IA/IE-negative group exhibited a decreasingtrend, and the IA/IE-positive group was significantly decreased (t test;significance level *; P<0.05). In conclusion, the mouse CT26intratumoral monocyte/macrophage population or a portion of thepopulation was found to have a decreased intratumoral cell count.

Example 12 Evaluation of Antitumor Effect of Anti-mCCR8 AntibodyAdministration Using Colorectal Cancer-Derived CT26

3×10⁵ colorectal cancer-derived CT26 cells (50 uL) were intracutaneouslytransplanted to the back of each Balb/c mouse (7 weeks old, female). 3days after tumor inoculation, 400 μg (400 μL) of a rat anti-mouse CD198(CCR8) antibody (clone SA214G2, BioLegend, Inc.) was intravenouslyadministered thereto (N=10). An isotype control antibody wasadministered to a control (N=10). Tumor volumes were measured every 3 to4 days from 8 days after tumor inoculation (5 days after antibodyadministration). The tumor volume (mm³) was calculated according tomajor axis (mm)×minor axis (mm)×minor axis (mm)/2 (FIG. 19).

As a result, no significant difference was observed in the anti-mCCR8administration group compared with the isotype control antibodyadministration group at post-transplant day 7, whereas the tumor volumeof the anti-mCCR8 antibody administration group was significantlydecreased at 11, 14, 17 and 21 days after tumor inoculation(significance level: ***; P<0.001 at days 11 and 14, **; P<0.01 at days17 and 21). Furthermore, in the anti-mouse CCR8 antibody administrationgroup, the tumor volume was decreased at post-transplant day 14 orlater, and the tumors disappeared almost completely at day 17(individual-based data is shown in FIG. 20, and mean data is shown inFIG. 21). From these results, it was concluded that the anti-mCCR8antibody administration suppressed the functions of mCCR8 expressed onTreg and monocytes/macrophages pointed out as immunosuppressive cells,or killed (removed) these expressing cells through the ADCC activity ofthe antibody so that tumor immunity was enhanced, leading to theregression and disappearance of the tumors.

As already reported by many literatures, etc., in the case ofadministering an antibody specific for mouse CD25 (anti-CD25), a markerof mouse Treg cells, to mice and thereby removing mouse Treg cells, theadministration before tumor inoculation exhibits a weak antitumor effectand the administration at post-transplant day 2 or later exhibits noantitumor effect. We also carried out the administration of theanti-CD25 antibody at post-transplant day 3 using the same CT26 cellsystem as that used this time, but observed no antitumor effect. Fromthese results, it was concluded that the anti-mCCR8 antibody hasstronger drug efficacy than that of the anti-CD25 antibody.

Example 13

Next, anti-PD-1 (clone RMP1-14, Bio X Cell), an antibody specific formouse PD-1, was evaluated for its drug efficacy using CT26 andcomparatively studied with anti-mCCR8. 2×10⁵ colorectal cancer-derivedCT26 cells (50 μL) were intracutaneously transplanted to the back ofeach Balb/c mouse (7 weeks old, female). The anti-PD-1 antibody (200μg/head, i.p.) was administered a total of three times every 3 to 4 daysfrom post-transplant day 7.

As a result, an antitumor effect was observed in the group given theanti-PD-1 antibody (N=8) compared with a group given an isotype controlantibody (N=8). The mean tumor volume and standard deviation of theisotype control were 601.7±378.1 mm³, 956.3±467.7 mm³ and 1528.4±774.1mm³ at 14, 17 and 20 days after tumor inoculation, respectively, whilethe mean tumor volume and standard deviation of the anti-PD-1 antibodyadministration group were 175.3±42.6 mm³, 174.7±55.8 mg and 209.6±99.8mm³ at 14, 17 and 20 days after tumor inoculation, respectively. Theanti-PD-1 antibody significantly suppressed increase in tumor volume ascompared with the control at all of 14, 17 and 20 days after tumorinoculation. However, an individual whose tumor disappeared completelywas 1 out of 8 mice in the observation period (up to post-transplant day20). On the other hand, the complete disappearance of tumors wasobserved in all of 10 cases in the same period as above by anti-mCCR8antibody administration. From these results, it was concluded that theanti-mCCR8 antibody has stronger drug efficacy than that of theanti-PD-1 antibody in the standard administration method.

Example 14 Confirmation of Presence or Absence of Induction ofAutoimmune Disease in Mouse Given Anti-mCCR8 Antibody

Next, the states of the mice of Example 12 were evaluated up topost-administration day 18. No significant difference in body weight inthis period was found between the control antibody administration groupand the anti-CCR8 antibody administration group. Piloerection was notobserved in both the groups. These mice were dissected atpost-administration day 18. Although the presence or absence ofenlargement of the lymph node and the intestinal tract was studied inthe anti-CCR8 administration group compared with the control, theenlargement was not observed without any difference between the groups.From these findings, it was concluded that any sign of autoimmunedisease was not observed in the period when an antitumor effect wasexerted on the mice given the anti-CCR8 antibody. Papers have reportedthat, in general, if Treg is removed from the whole body of a mouse tothe extent that an antitumor effect is induced, severe autoimmunedisease is induced around 14 days after removal. This is a matter ofconcern for tumor immunotherapy including Treg suppression therapy. Theresults obtained this time showed that any autoimmune disease was notinduced even at post-antibody administration day 18 in the mice in whicha strong anti-tumor immunity effect was observed by anti-CCR8 antibodyadministration. One of the explanations therefor is the low expressionof mouse and human CCR8 in PBMCs, the spleen, and the lymph nodecompared with tumor tissues according to reports. However, none of theprevious reports state whether or not an autoimmune disease is inducedby removing or functionally inhibiting CCR8-expressing Treg cells inthese peripheral tissues. Here, it was found for the first time thatthese approaches induce no autoimmune disease. This effect may beunexpectable from the previous findings.

Example 15 Evaluation of Antitumor Effect of Anti-mCCR8 AntibodyAdministration Using Colorectal Cancer-Derived Colon-26

2×10⁵ colorectal cancer-derived Colon-26 cells (50 μL) wereintracutaneously transplanted to the back of each Balb/c mouse (7 weeksold, female). 3 days after tumor inoculation, 400 μg (400 μL) of a ratanti-mouse CCR8 antibody (clone SA214G2, BioLegend, Inc.) wasintravenously administered thereto (N=10). An isotype control antibodywas administered to a control (N=10). Tumor volumes were measured every3 to 4 days from 3 days after tumor inoculation (5 days after antibodyadministration). The tumor volume (mm³) was calculated according tomajor axis (mm)×minor axis (mm)×minor axis (mm)/2. The point in timewhen the tumor reached an endpoint volume (800 mm³) was used as theendpoint of each animal. As a result, increase in tumor volume wassuppressed in the anti-mCCR8 administration group compared with theisotype control antibody administration group at 14 and 18 days aftertumor inoculation. The mean tumor volume at day 14 was 451.3 mm³(standard deviation: ±177.5 mm³) in the isotype control antibodyadministration group and 322.6 mm³ (standard deviation: ±146.0 mm³) inthe anti-CCR8 antibody administration group. An individual having atumor volume of 350 mm³ or larger at day 14 was 9 out of 10 cases in theisotype control group and 4 out of 10 cases in the anti-mCCR8administration group. There was a significant difference with P=0.019 inthe Pearson's chi-square test as to this segregated form. Thus, thedifference was observed in the number of individuals whose tumor volumereached 350 mm³ at day 14. Also, the mean tumor volume atpost-transplant day 18 was 874.7 mm³ (standard deviation: ±269.2 mm³) inthe isotype control antibody administration group and 585.4 mm³(standard deviation: ±401.7 mm³) in the anti-CCR8 antibodyadministration group (FIG. 22). An individual having a tumor volume of600 mm³ or larger at day 18 was 9 out of 10 cases in the isotype controlgroup and, on the other hand, 4 out of 10 cases in the anti-mCCR8administration group. There was a significant difference with P=0.019 inthe Pearson's chi-square test as to this segregated form. Thus, thedifference was observed in the number of individuals whose tumor volumereached 600 mm³ at day 18. Further, the point in time when the tumorvolume reached 800 mm³ was preset as an endpoint. An individual regardedas being dead with the tumor volume exceeding 800 mm³ was observed inneither of the groups up to day 14 and was 7 out of 10 cases in theisotype control group and 3 out of 10 cases in the anti-CCR8 antibodygroup at day 18. As a result of studying a difference in the survivalprobability at day 18 by the Pearson's chi-square test, there was asignificant difference in the survival probability with P=0.025.

No antitumor effect was observed in an anti-PD-1 (clone RMP1-14, Bio XCell) administration group compared with a group given an isotypecontrol antibody in a similar experiment using the same cell line asabove. In conclusion, the anti-mCCR8 antibody exhibited a higherantitumor effect on anti-PD-1 antibody-resistant Colon26 cells.

Example 16 Analysis on Expression of CCR8 in Human Kidney CancerInfiltrating Cells

The expression of CCR8 was analyzed in human kidney cancertumor-infiltrating cells of 14 cases. The backgrounds of the 14 kidneycancer patients were 11 males and 3 females for sex, a median age of68.5 years, and pathological stages of T1A for 6 patients, T1B for 2patients, T3A for 5 patients, and T3b for 1 patient. Specifically,kidney cancer primary tumor-infiltrating cells were isolated from the 14kidney cancer (clear cell renal cell carcinoma, ccRCC) patients in thesame way as in FIG. 1 of Example 1, stained with anti-CD4 (BioLegend,Inc., Clone OKT4), anti-CD3 (BioLegend, Inc., Clone UCHT1), anti-CD45RA(BD Biosciences, Clone HI100), anti-CD8 (BioLegend, Inc., RPA-T8),anti-CCR8 (BioLegend, Inc., Clone L263G8), and anti-FoxP3 (eBioscience,Inc., Clone 236A/E7) or an anti-FoxP3 isotype control antibody, andanalyzed by flow cytometry (BD Biosciences, BD LSRFortessa). CD3+CD8+ Tcells and CD3+CD4+ T cells were analyzed. The CD3+CD4+ T cells werefurther divided into 2 groups according to the presence or absence ofFoxP3 expression and analyzed. A FoxP3 expression-negative control wasprepared by staining with the isotype control antibody. The mean valueof FACS analysis (MFI) of each patient sample was used as the expressionintensity of CCR8. Table 1 shows mean MFI of staining with the anti-CCR8antibody or the isotype control antibody thereof, and a standarddeviation thereof.

TABLE 1 Cell CD8+ T FoxP3− CD4+ T FoxP3+ CD4+ T Antibody Anti- Anti-Anti- Isotype mCCR8 Isotype mCCR8 Isotype mCCR8 Mean MFI 84.9 267 56.9423 131.3 3507.2 Standard 26.8 159 62.1 297.5 59 1466.3 deviation

The CD8+ T cells were found to rarely express CCR8 (Table 1). The CD4+FoxP3− T cells slightly expressed CCR8, whereas the CD4+ FoxP3+ T cellshad 8 times or more the mean MFI of the CD4+ FoxP3− T cells, revealingthat the CD4+ FoxP3+ T cells significantly strongly express CCR8 (Table1). FIG. 23 shows the results of Table 1 in a graph form. Each plot ofthe graph shows the mean CCR8 expression level (MFI) of each patientsample in a flow cytometer. The horizontal lines of the graph depict themean MFI of the samples. The bars depict standard deviations.Significance level *** denotes P<0.001. From these results, the CCR8protein was found to be specifically expressed on the surface ofCD3+CD4+ FoxP3+ T cells which infiltrate tumors in human kidney cancer(ccRCC). These results are also consistent with the results of the mRNAexpression analysis by the RNA-Seq analysis.

Tumor-infiltrating CD4+ T cells in the 14 ccRCC samples described abovewere subjected to flow cytometry analysis using FoxP3 and CCR8. Theratio of CCR8-positive cells to FoxP3-positive cells and the ratio ofCCR8-positive cells to FoxP3-negative cells were plotted on a samplebasis (FIG. 24). Staining with an isotype control antibody was used asnegative standards for both FoxP3 and CCR8, and cells having a valueequal to or more than this threshold were used as positive cells. As aresult, the CCR8 expression rate of intratumoral CD3+CD4+ FoxP3+ T cellswas approximately 75%, and the CCR8 expression rate of CD3+CD4+ FoxP3− Tcells was approximately 10%.

From these results, CCR8 was found to be expressed in most ofFoxP3-expressing Treg cells among human kidney cancer tumor-infiltratingcells and expressed in approximately 10% of CD4-positive T cells otherthan the Treg cells. From these results, the CCR8 expression rate ofhuman intratumoral FoxP3-positive Treg cells was similar to that ofmouse intratumoral Treg cells, indicating the possibility that theanti-human CCR8-specific antibody can remove most of tumor-infiltratingFoxP3-positive Treg cells, as in mice.

Example 17

Correlation of CCR8 Expression Rate of Tumor-Infiltrating Cells inVarious Cancers with Survival Probability

FoxP3 gene has been identified as a gene that is specifically expressedin Treg cells and not expressed in tumor cells or most of normal humancells. For example, FoxP3 gene as a marker gene of Treg cells, CD3G geneas a marker gene of T cells and NK cells, and CD8A gene as a marker geneof CD8-positive T cells are known as so-called marker genes, which areexpressed only in certain specific cells as mentioned above.

It has also been reported as to the FoxP3 gene, a marker gene of Tregcells, that the mRNA expression level of the FoxP3 gene within eachtumor may be measured and thereby used as an index for the abundanceratio of Treg cells within the tumor (Cell, 2015, Vol. 160, p. 48-61).

As also reported in this paper, whether the intratumoral abundance ratioof Treg cells is related to a survival probability may be analyzed bydrawing a Kaplan-Meier survival curve as to the intratumoral expressionrate (Treg abundance ratio) of the marker gene and patients' survivalprobabilities through the use of a RNA-Seq database such as TCGA. TheRNA-Seq data on tumor masses is mixed data on mRNA expressed in bothtumor cells and infiltrating cells present therein (lymphocytes,vascular cells, etc.). However, a gene shown to be not expressed intumor cells can be regarded as a gene expressed in tumor-infiltratingcells. Through the use thereof, the tumor-infiltrating cells can beidentified by the analysis as described above, i.e., analysis on markergene expression using the RNA-Seq data on tumor masses. Furthermore, theexpression level of a marker gene in a tumor mass can be regarded as theproduct of an expressing cell count of particular cells, correspondingto the marker gene, infiltrating the tumor mass, and the expressionlevel of the marker gene in each expressing cell.

In this context, if the expression level of the marker gene in each cellis almost constant among individuals, the expression level is in directproportion to an infiltrating cell count. Thus, an intratumoralexpressing cell count can be calculated on an individual basis by use ofthis expression level and can be compared among individuals.

(CCR8 Expression Analysis at Cell Level)

RNA expression data from 1037 different types of human cell lines isregistered in a public database CCLE (Cancer Cell Line Encyclopedia).Whether CCR8 or CD3G gene would be expressed in cancer cells other thanT cells or normal cells was analyzed using the database.

The mRNA expression of CD3G and CCR8 was analyzed as to kidney cancer-,prostate cancer- and bladder cancer-derived cell lines using the CCLEdatabase.

The cell lines examined were 40 kidney cancer-derived cell lines;VMRCRCW, SKRC20, SNU34, SKRC31, UOK10, SLR20, OSRC2, TUHR14TKB, SLR24,HK2, A498, RCC4, KMRC1, RCC10RGB, ACHN, SLR25, SNU1272, UMRC6, SLR23,769P, SLR21, HEKTE, CAKI1, TUHR4TKB, KMRC2, VMRCRCZ, KMRC3, KMRC20,CAKI2, BFTC909, 7860, A704, TUHR10TKB, SLR26, UMRC2, CAL54, FURPNT1,FURPNT2, HEK293, and G402; 8 prostate cancer-derived cell lines; VCAP,LNCAPCLONEFGC, DU145, PC3, 22RV1, PRECLH, MDAPCA2B, and NCIH660; and 2bladder cancer-derived cell lines; TCBC14TK and TCBC2TKB. In all ofthese solid cancer cell lines examined, the expression of CCR8 and CD3Gwas at the same level as the background level, and no mRNA expressionwas observed (even the largest value indicating expression was 1/500 orless of the expression level of G3PDH, and all the other values were1/1000 or less of the expression level of G3PDH). In short, CCR8 andCD3G were able to be confirmed to be rarely expressed on solid cancercells. Primary normal cells derived from each human tissue were alsoanalyzed in the same way as above. CCR8 and CD3G were found to beexpressed only in some hematopoietic cells and rarely expressed in theother tissues-derived primary normal cells.

These results showed that the cells of these 3 cancers express neitherCCR8 nor CD3G. Thus, it was concluded that TCGA RNA expression data usedfor tumor masses of kidney cancer, prostate cancer and bladder cancerreflects the mRNA expression of CCR8 and CD3G in infiltrating normalcells, other than cancer cells, present in the tumor masses.

(Analysis Using Public TCGA Database)

Next, the ratio of the CCR8 gene to the CD3G gene (CCR8/CD3G) expressedin the tumor of kidney cancer, prostate cancer, or bladder cancer, andpatients' survival probabilities were analyzed through the use of thepublic TCGA database. A gene that most highly correlated (Pearson'scorrelation) in terms of expression with the CCR8 and CD3G genes withinthese 3 tumors was found to be various genes specifically expressed in Tcells (FoxP3, CD5, IL7R, etc. with correlation coefficient r of 0.7 ormore). These results indicate that CCR8 or CD3G is not expressed ontumor cells themselves and is specifically expressed ontumor-infiltrating expressing cells (particularly, T cells). However, aCCR8-expressing cell population was used here because this does not denythat CCR8 is expressed on infiltrating cells other than T cells. CD3G,as already reported in papers, etc., is specifically expressed on Tcells and NK cells. Also, T cells are major tumor-infiltrating cells.Therefore, an infiltrating T cell count can be hypothesized from a CD3Gexpression level. Thus, the CCR8/CD3G value can be defined as aCCR8-expressing cell count per T cell count present within a tumor.

The CCR8/CD3G ratio and patients' survival probabilities were analyzedas to these 3 carcinomas using a Kaplan-Meier curve. For kidney cancer,Kidney Renal Clear Cell Carcinoma (TCGA, Provisional) data in the TCGAdata was used, and 523 cases having complete RNA expression data andpatients' survival probability data were used. Likewise, for prostatecancer, Prostate Adenocarcinoma (TCGA, Provisional) data in the TCGAdata was used, and 490 cases having complete RNA expression data andpatients' survival probability data were used.

Also, for bladder cancer, Bladder Urothelial Carcinoma (TCGA,Provisional) data in the TCGA data was used, and 392 cases havingcomplete RNA expression data and patients' survival probability datawere used.

Patients of each cancer were equally divided into 2 groups (the kidneycancer patients were odd-numbered and therefore divided into 261:262)with high CCR8/CD3G expression values and with low CCR8/CD3G expressionvalues, followed by Kaplan-Meier survival curve analysis usinganalytical software R (R-Studio). The log-rank test was conducted as asignificant difference test. The results about the kidney cancer areshown in FIG. 25, the results about the prostate cancer are shown inFIG. 26, and the results about the bladder cancer are shown in FIG. 27.The vertical lines in the graphs show that the patients survived butwere treated as dropouts (corresponding to so-called censors) at thepoint in this time because the evaluation period was terminated at thispoint in time. The values on the abscissa depict the number of months inall the graphs.

As a result, in all the 3 carcinomas, the groups with high CCR8/CD3Gvalues had significantly low patients' survival probabilities. Thegroups with a high ratio of human tumor-infiltrating CCR8-expressingcells to T cells were found to have a reduced survival probability. Thissuggests that in humans as well, CCR8-expressing cells have asuppressive effect on tumor immunity. This suggests the possibilitythat, as in the antitumor effect of the anti-mCCR8 antibody administeredto mice, intratumoral CCR8-expressing cells in humans are specificallyremoved or killed by some method to thereby enhance tumor immunity andelevate a survival probability.

Example 18 Confirmation of Expression of Mouse CCR8 in LM8 Cells andMethA Cells

Osteosarcoma-derived LM8 cells or skin fibrosarcoma-derived MethA cellswere cultured in a 6-well dish, and the culture solution was removedwhen the cells became approximately 50% confluent. 5 ml of 10 mMEDTA/PBS was added thereto, and the cells were incubated at 37° C. for 5minutes. As a result, almost all the cells were dissociated, suspendedusing a pipette and were thereby able to be separated into almost singlecells. The cells were washed twice with D-MEM/10% FCS, suspended inD-MEM/10% FCS, and stained in ice with LIVE/DEAD® Fixable Near-IR DeadCell Stain Kit (Thermo Fisher Scientific Inc., L34975) and an anti-mouseCCR8 (SA214G2) or isotype control antibody. 1 hour later, the cells werewashed three times with D-MEM/10% FCS and analyzed for a mouse CCR8expression rate using a flow cytometer (FACSCanto II). A background wasset using the isotype control antibody, and the proportion of positivecells equal to or larger than the background level and medianfluorescence were calculated (FIG. 28). As a result, no difference inmedian PE fluorescence intensity was observed in both the cells, and thepositive cells were not observed. In conclusion, these cells were notrecognized by the anti-mouse CCR8 antibody and were confirmed to neitherexpress mouse CCR8 nor retain an epitope reactive with the antibody.

Example 19 Evaluation of Antitumor Effect of Anti-Mouse CCR8 AntibodyAdministration Using Osteosarcoma-Derived LM8

3×10⁵ mouse osteosarcoma-derived LM8 cells (50 uL) were intracutaneouslytransplanted to the back of each C3H/He mouse (7 weeks old, male). 3days after tumor inoculation, 400 μg (400 μL) of a rat anti-mouse CCR8antibody (clone SA214G2, BioLegend, Inc.) was intraperitoneallyadministered thereto (N=11). An isotype control antibody wasadministered to a control (N=10). Tumor volumes were measured every 3 to4 days from 7 days after tumor inoculation (4 days after antibodyadministration). The tumor volume (mm³) was calculated according tomajor axis (mm)×minor axis (mm)×minor axis (mm)/2 (FIG. 29). As aresult, the mean tumor volume of the anti-mCCR8 administration groupcompared with the isotype control antibody administration group wassignificantly decreased at all the points in time of measurement atpost-transplant day 18 or later (significance level: *; P<0.05 at day18, **; P<0.01 at days 21, 24, 27 and 31, ***; P<0.001 at day 35).Furthermore, the tumors disappeared in 6 out of 11 mice in theanti-mouse CCR8 antibody administration group and 1 out of 10 mice inthe isotype control antibody administration group at post-antibodyadministration day 31. There was a significant difference (P=0.031) inthe Pearson's chi-square test conducted as to this segregated form.

Example 20 Evaluation of Antitumor Effect of Anti-Mouse CCR8 AntibodyAdministration Using Skin Fibrosarcoma-Derived MethA

1×10⁵ skin fibrosarcoma-derived MethA cells (50 uL) wereintracutaneously transplanted to the back of each Balb/c mouse (7 weeksold, female). 3 days after tumor inoculation, 400 μg (400 μL) of a ratanti-mouse CCR8 antibody (clone SA214G2, BioLegend, Inc.) wasintraperitoneally administered thereto (N=5). An isotype controlantibody was administered to a control (N=5). Tumor volumes weremeasured every 3 to 4 days from 11 days after tumor inoculation (8 daysafter antibody administration). The tumor volume (mm³) was calculatedaccording to major axis (mm)×minor axis (mm)×minor axis (mm)/2 (FIG.30).

As a result, the mean tumor volume of the anti-mouse CCR8 administrationgroup compared with the isotype control antibody administration groupwas significantly decreased at all the points in time of measurement atpost-transplant day 11 or later (significance level: *; P<0.05 at allthe points in time). Furthermore, the tumors disappeared in 5 out of 5mice in the anti-mouse CCR8 antibody administration group and 0 out of 5mice in the isotype control antibody administration group atpost-antibody administration day 21. There was a significant difference(P=0.0016) in the Pearson's chi-square test conducted as to thissegregated form.

Example 21 Evaluation of Antitumor Effect of Anti-Mouse CCR8 AntibodyAdministration Using Breast Cancer-Derived EMT6

1×10⁵ breast cancer-derived EMT6 cells (50 uL) were intracutaneouslytransplanted to the back of each Balb/c mouse (7 weeks old, female). 3and 10 days after tumor inoculation, 100 μg (100 μL) of a rat anti-mouseCCR8 antibody (clone SA214G2, BioLegend, Inc.) was intraperitoneallyadministered thereto (N=20). An isotype control antibody wasadministered to a control (N=20). Tumor volumes were measured every 3 to4 days from 4 days after tumor inoculation (1 day after antibodyadministration). The tumor volume (mm³) was calculated according tomajor axis (mm)×minor axis (mm)×minor axis (mm)/2 (FIG. 31).

As a result, the mean tumor volume of the anti-mouse CCR8 administrationgroup compared with the isotype control antibody administration groupwas significantly decreased at all the points in time of measurement atpost-transplant day 10 or later (significance level: **; P<0.01 at day10, ***; P<0.001 at days 14, 17 and 21). Furthermore, the tumorsdisappeared in 19 out of 20 mice in the anti-mouse CCR8 antibodyadministration group and 2 out of 20 mice in the isotype controlantibody administration group at post-antibody administration day 21.There was a significant difference (P<0.0001) in the Pearson'schi-square test conducted as to this segregated form.

Example 22 Confirmation of Superiority of Anti-Mouse CCR8 Antibody OverAnti-PD-1 Antibody

2×10⁵ colorectal cancer-derived Colon26 cells (50 uL) wereintracutaneously transplanted to the back of each Balb/c mouse (7 weeksold, female). 3 and 10 days after tumor inoculation, 400 μg (400 μL) ofan isotype control antibody, a rat anti-mouse CCR8 antibody (cloneSA214G2, BioLegend, Inc.) or an anti-mouse PD-1 antibody (RMP1-14, Bio XCell) was intravenously administered thereto (N=10). Tumor volumes weremeasured every 3 to 4 days from 3 days after tumor inoculation. Thetumor volume (mm³) was calculated according to major axis (mm)×minoraxis (mm)×minor axis (mm)/2 (FIG. 32). As a result, the tumor volume ofthe anti-mouse CCR8 administration group compared with the isotypeantibody administration group was significantly decreased at days 17,20, and 24 (Steel's nonparametric test: significance level P<0.05). Nosignificant difference was observed in the anti-PD-1 antibodyadministration group compared with the isotype antibody administrationgroup at any point in time.

A mouse individual bearing a tumor with a volume of 1000 mm3 or largerat post-antibody administration day 24 was 7 out of 10 mice in theisotype antibody administration group, 2 out of 10 mice in theanti-mouse CCR8 antibody administration group, and 7 out of 10 mice inthe anti-PD-1 administration group. The anti-CCR8 administration grouphad a significant difference from both the isotype antibodyadministration group and the anti-PD-1 antibody administration group inthe Pearson's chi-square test as to the segregated form (P=0.025 forboth). In conclusion, the anti-mouse CCR8 antibody administration wasconfirmed to produce an antitumor therapeutic effect on the colorectalcancer cell line Colon26.

Furthermore, the tumor volume of the anti-mouse CCR8 administrationgroup compared with the anti-mouse PD-1 antibody administration groupwas significantly decreased at 20 and 24 days after tumor inoculation(Steel-Dwass nonparametric test; significance level P<0.05). Inconclusion, a stronger antitumor therapeutic effect on the mousecolorectal cell line was observed in the anti-mouse CCR8 antibodyadministration group compared with the anti-PD-1 antibody administrationgroup.

Example 23 Evaluation of Antitumor Effect of Anti-Mouse CCR8 AntibodyAdministration Using Kidney Cancer-Derived Cell Line RAG

A similar study was conducted using a mouse kidney cancer-derived cellline RAG. 4×10⁵ kidney cancer-derived RAG cells (50 uL) wereintracutaneously transplanted to the back of each Balb/c mouse (8 weeksold, female). 6 days after tumor inoculation, 100 μg (100 μL) of anisotype control antibody (N=10 except for N=9 at day 21), a ratanti-mouse CCR8 antibody (N=10) (clone SA214G2, BioLegend, Inc.) or ananti-mouse PD-1 antibody (N=10) (RMP1-14, Bio X Cell) wasintraperitoneally administered thereto. Tumor volumes were measuredevery 3 to 4 days from 6 days after tumor inoculation. The tumor volume(mm³) was calculated according to major axis (mm)×minor axis (mm)×minoraxis (mm)/2 (FIG. 33). As a result, the tumor volume of the anti-mouseCCR8 administration group compared with the isotype antibodyadministration group was significantly decreased at 14, 17, and 21 daysafter tumor inoculation (Steel's nonparametric test: significance levelP<0.05). No significant difference was observed in the anti-mouse PD-1antibody administration group compared with the isotype antibodyadministration group. In conclusion, the anti-mouse CCR8 antibodyadministration was confirmed to produce an antitumor therapeutic effecton the kidney cancer cell line. Furthermore, the tumor volume of theanti-mouse CCR8 administration group compared with the anti-mouse PD-1antibody administration group was significantly decreased atpost-transplant day 14 (Steel-Dwass nonparametric test; significancelevel P<0.05). In conclusion, a stronger antitumor therapeutic effect onthe mouse kidney cancer cell line was observed in the anti-mouse CCR8antibody administration group compared with the anti-mouse PD-1 antibodyadministration group.

Example 24 Analysis on Presence or Absence of Inflammatory Response inMouse Given Anti-Mouse CCR8

2×10⁵ colorectal cancer-derived Colon26 cells (50 uL) wereintracutaneously transplanted to the back of each Balb/c mouse (7 weeksold, female). 3 and 10 days after tumor inoculation, 400 μg (400 μL) ofa rat anti-mouse CD198 (CCR8) antibody (clone SA214G2, BioLegend, Inc.)or an isotype control antibody (LTF-2, Bio X Cell) was intravenouslyadministered thereto (N=10). The body weight and the weight of eachmouse organ (lung, liver, spleen, small intestine, and inguinal node)were measured at post-transplant day 24 (FIG. 34). As a result, as shownin FIG. 34, no significant difference in body weight and each organweight was observed between the control administration group (N=10) andthe anti-mouse CCR8 antibody administration group (N=10). From theseresults, it was concluded that the anti-mouse CCR8 antibodyadministration induced neither inflammatory response nor an autoimmunedisease.

Example 25 Analysis on Expression of CCR8 in Various ClinicalTumor-Infiltrating Cells

The expression of CCR8 was analyzed in tumor-infiltrating cells of humankidney cancer, ovary cancer, uterine corpus cancer, colorectal cancer,and lung cancer. The numbers of patients with various clinical tumorsused in the expression analysis were 12 kidney cancer patients, 14 ovarycancer patients, 21 uterine corpus cancer patients, 10 colorectal cancerpatients, and 4 lung cancer patients. Various clinicaltumor-infiltrating cells were isolated in the same way as in FIG. 1 ofExample 1 and stained with anti-CD45 (BioLegend, Inc., Clone H130) andanti-CCR8 (BioLegend, Inc., Clone L263G8) antibodies, followed bymeasurement by flow cytometry (BD Biosciences, BD LSRFortessa). ACCR8-positive cell count per tumor weight and the ratio of CCR8-positivecells to CD45-positive leukocytes were analyzed.

Table 2 shows a mean CCR8-positive cell count per tumor weight and astandard deviation thereof. Table 3 shows a mean ratio of CCR8-positivecells to CD45-positive leukocytes and a standard deviation thereof.

TABLE 2 CCR8-positive cell count (×10⁵) per tumor weight (g) Cancer typeMean Standard deviation Kidney cancer 8.9 22.7 Ovary cancer 1.7 2.6Uterine corpus cancer 13.1 28.5 Colorectal cancer 2.9 5.4 Lung cancer21.8 36.9

TABLE 3 Ratio (%) of CCR8-positive cells to CD45- positive leukocytesCancer type Mean Standard deviation Kidney cancer 5.6 5.2 Ovary cancer5.2 6.6 Uterine corpus cancer 9.0 9.2 Colorectal cancer 6.2 6.5 Lungcancer 2.9 2.3

In the various clinical tumors with kidney cancer as a reference, as forthe CCR8-positive cell count per tumor weight, ovary cancer andcolorectal cancer exhibited a lower mean than that of kidney cancer, anduterine corpus cancer and lung cancer exhibited a higher mean than thatof kidney cancer. As for the ratio of CCR8-positive cells toCD45-positive leukocytes, ovary cancer exhibited a mean equivalent tothat of kidney cancer, and lung cancer exhibited a lower mean than thatof kidney cancer. Also, uterine corpus cancer and colorectal cancerexhibited a higher mean than that of kidney cancer. The expression ofCCR8 was confirmed in the tumor-infiltrating cells of ovary cancer,uterine corpus cancer, colorectal cancer and lung cancer, in addition tothe human kidney cancer tumor-infiltrating cells. These resultsindicated the possibility that in kidney cancer as well as ovary cancer,uterine corpus cancer, colorectal cancer and lung cancer, CCR8-positivetumor-infiltrating cells can be removed using the anti-humanCCR8-specific antibody.

Example 26 Evaluation of Antitumor Effect of Combined Administration ofAnti-Mouse CCR8 Antibody and Anti-PD-1 Antibody Using BreastCancer-Derived EMT6

1×10⁵ breast cancer-derived EMT6 cells (50 uL) were intracutaneouslytransplanted to the back of each Balb/c mouse (7 weeks old, female).

To an anti-mouse CCR8 antibody alone administration group, 15 μg of arat anti-mouse CCR8 antibody (clone SA214G2, BioLegend, Inc.) wasintravenously administered (100 μL) 3 and 10 days after tumorinoculation, and 200 μg (100 μL) of an isotype control antibody wasadministered at 8 and 13 days after tumor inoculation (N=10). To ananti-PD-1 antibody alone administration group, 15 μg (100 μL) of anisotype control antibody was intravenously administered 3 and 10 daysafter tumor inoculation, and 200 μg (100 μL) of an anti-mouse PD-1antibody (RMP1-14, Bio X Cell) was intravenously administered at 8 and13 days after tumor inoculation (N=10). To an anti-PD-1 antibody andanti-mouse CCR8 antibody combined administration group, 15 μg (100 μL)of the rat anti-mouse CCR8 antibody was intravenously administered 3 and10 days after tumor inoculation, and 200 μg (100 μL) of the anti-PD-1antibody was intravenously administered at 8 and 13 days after tumorinoculation (N=10). To a control group, 15 μg (100 μL) of an isotypecontrol antibody was intravenously administered 3 and 10 days aftertumor inoculation, and 100 μL. of PBS was intravenously administered at8 and 13 days after tumor inoculation (N=10). Tumor volumes weremeasured every 3 to 4 days from 3 days after tumor inoculation (1 dayafter antibody administration). The tumor volume (mm³) was calculatedaccording to major axis (mm)×minor axis (mm)×minor axis (mm)/2 (FIG.35).

In the comparison of a mean tumor volume between the aloneadministration groups, the mean tumor volume was significantly small inthe anti-mouse CCR8 antibody administration group compared with theanti-PD-1 antibody administration group at days 10, 14, 17, 20, 23 and27 (Dunnett method, significance level: P<0.05). Also, the tumors weresmall in the combined administration group compared with each aloneadministration group.

A complete remission rate of the tumors at 17 and 27 days after tumorinoculation was also compared. At post-transplant day 17, the completeremission of the tumors was exhibited in 0 out of 10 mice in the controlgroup and the anti-PD-1 antibody administration group and 1 out of 10mice in the anti-mouse CCR8 antibody administration group, whereas thetumors remitted completely in 6 out of 10 mice in the anti-PD-1 antibodyand anti-mouse CCR8 antibody combined administration group. Atpost-transplant day 27, the complete remission of the tumors wasexhibited in 2 and 3 out of 10 mice in the control group and theanti-PD-1 antibody administration group, respectively, and 7 out of 10mice in the anti-mouse CCR8 antibody administration group, whereas thetumors remitted completely in 9 out of 10 mice in the anti-PD-1 antibodyand anti-mouse CCR8 antibody combined administration group.

The proportion of an individual bearing tumor larger than 50 mm³ orsmaller was further calculated (FIG. 36). Tue tumors larger than 50 mm³or smaller in all the individuals in the anti-PD-1 antibody andanti-mouse CCR8 antibody combined administration group (100%) atpost-transplant day 17 and then were 50 mm³ or smaller up to day 27,whereas the proportion was 10% and 30% in the anti-PD-1 antibodyadministration group at days 17 and 27, respectively, and 70% in theanti-mouse CCR8 antibody administration group at both 17 and 27 daysafter tumor inoculation.

These results demonstrated that the combined administration grouprequires a short time to tumor regression and has a strong regressingeffect, as compared with other alone administration groups.

Example 27 Evaluation of Antitumor Effect of Combined Administration ofAnti-Mouse CCR8 Antibody and Anti-PD-1 Antibody Using Mouse KidneyCancer-Derived Cell Line RAG

4.5×10⁵ kidney cancer-derived RAG cells (50 uL) were intracutaneouslytransplanted to the back of each Balb/c mouse (6 weeks old, female). TheRAG cells used were RAG cells (acclimatized cell line) with mousesubcutaneous engraftment efficiency elevated by transplanting, again toa mouse, a tumor successfully engrafted in advance by subcutaneousinoculation to a Balb/c mouse and repeating this operation twice.

To an anti-PD-1 antibody alone administration group, 50 μg (100 μL) ofan anti-PD-1 antibody (RMP1-14, Bio X Cell) was intravenouslyadministered 8 and 15 days after tumor inoculation (N=10). To ananti-mouse CCR8 antibody alone administration group, 25 μg (100 μL) of arat anti-mouse CCR8 antibody (clone SA214G2, BioLegend, Inc.) wasintravenously administered 8 and 15 days after tumor inoculation (N=10).To an anti-PD-1 antibody and anti-mouse CCR8 antibody combinedadministration group, 50 μg of an anti-PD-1 antibody (RMP1-14, Bio XCell) and 25 μg of a rat anti-mouse CCR8 antibody (clone SA214G2,BioLegend, Inc.) were mixed (100 μL) and intravenously administered 8and 15 days after tumor inoculation (N=10). To a control group, 100 μLof physiological saline was intravenously administered 8 and 15 daysafter tumor inoculation (N=10).

Tumor volumes were measured every 3 to 4 days from 8 days after tumorinoculation. The tumor volume (mm³) was calculated according to majoraxis (mm)×minor axis (mm)×minor axis (mm)/2 (FIG. 37).

As a result, the tumors were found to be reduced in size in theanti-PD-1 antibody and anti-mouse CCR8 antibody combined administrationgroup compared with the anti-PD-1 antibody or anti-mouse CCR8 antibodyalone administration group.

Example 28 Analysis on Specificity of Anti-Mouse CCR8 Antibody UsingHomozygously CCR8 Gene-Deficient Mouse

3×10⁵ colorectal cancer-derived Colon26 cells (50 uL) wereintracutaneously transplanted to the back of each wild-type mouse (N=10)or homozygously CCR8 gene-deficient mouse (N=5) of Balb/c lineage. Tothe wild-type mouse, 100 μg (100 μL) of a rat anti-mouse CCR8 antibody(clone SA214G2, BioLegend, Inc.) or an isotype control antibody (LTF-2,Bio X Cell) was intravenously administered 3 and 10 days after tumorinoculation (N=5). To the homozygously CCR8 gene-deficient mouse, 100 μg(100 μL) of a rat anti-mouse CCR8 antibody (clone SA214G2, BioLegend,Inc.) or an isotype control antibody (LTF-2, Bio X Cell) was alsointravenously administered 3 and 10 days after tumor inoculation (N=5).Tumor sizes were measured from post-administration day 7.

As a result, significant tumor regression and final complete tumorregression were observed in all the wild-type mice by the anti-mouseCCR8 antibody administration compared with the isotype control antibodyadministration. On the other hand, neither change in tumor volume nortumor regression was observed in the homozygously CCR8 gene-deficientmice in the anti-mouse CCR8 antibody administration group compared withthe isotype antibody administration group (FIG. 38).

The antitumor effect of the anti-mouse CCR8 antibody disappearedcompletely in the homozygously CCR8 gene-deficient mice, demonstratingthat the anti-mouse CCR8 antibody (SA214G2) used exerts an antitumoreffect via CCR8.

INDUSTRIAL APPLICABILITY

The antibody against CCR8 of the present invention has an effect ofactivating the immunity by decreasing the number of tumor-infiltratingTreg cells or the like and is thus pharmaceutically useful for thetreatment of cancers.

1. A pharmaceutical composition for cancer treatment, comprising anantibody against CCR8.
 2. The pharmaceutical composition according toclaim 1, wherein the antibody against CCR8 is an antibody having ADCCactivity.
 3. The pharmaceutical composition according to claim 1,wherein the antibody against CCR8 is a CCR8-neutralizing antibody. 4.The pharmaceutical composition according to claim 1, wherein theantibody against CCR8 has an effect of removing tumor-infiltrating Tregcells.
 5. The pharmaceutical composition according to claim 1, whereinthe antibody against CCR8 has an effect of removing tumor-infiltratingmacrophage cells.
 6. The pharmaceutical composition according to claim1, wherein the cancer is breast cancer, colorectal cancer, kidney canceror sarcoma.
 7. A medicament for cancer treatment, comprising acombination of an antibody against CCR8 and an anti-PD-1 antibody or ananti-PD-L1 antibody.
 8. A method for treating a cancer, comprisingadministering an antibody against CCR8 according to claim
 1. 9. Theantibody against CCR8 according to claim 1 for treating a cancer.